U.S. patent number 6,096,504 [Application Number 09/248,335] was granted by the patent office on 2000-08-01 for maize glutathione-s-transferase enzymes.
This patent grant is currently assigned to E. I. du Pont Nemours and Company. Invention is credited to Brian McGonigle, Daniel P. O'Keefe.
United States Patent |
6,096,504 |
McGonigle , et al. |
August 1, 2000 |
Maize glutathione-S-transferase enzymes
Abstract
This invention relates to isolated nucleic acid fragments
encoding all or a substantial portion of maize
glutathione-S-transferase (GST) enzymes involved in the
detoxification of xenobiotic compounds in plants and seeds. The
invention also relates to the construction of chimeric genes
encoding all or a substantial portion of maize GST enzymes, host
cells transformed with those genes and methods of the recombinant
production of maize GST enzymes. Methods of constructing transgenic
plants having altered levels of GST enzymes and screens for
identifying maize GST enzyme substrates and maize GST enzyme
inhibitor, are also provided.
Inventors: |
McGonigle; Brian (Wilmington,
DE), O'Keefe; Daniel P. (Ridley Park, PA) |
Assignee: |
E. I. du Pont Nemours and
Company (Wilmington, DE)
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Family
ID: |
22938668 |
Appl.
No.: |
09/248,335 |
Filed: |
February 10, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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924759 |
Sep 5, 1997 |
5962229 |
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Current U.S.
Class: |
435/6.12;
435/193; 435/252.33; 435/320.1; 435/410; 435/455; 506/4; 536/23.1;
536/23.2; 536/23.6 |
Current CPC
Class: |
C12N
15/8274 (20130101); C12N 9/1088 (20130101) |
Current International
Class: |
C12N
15/82 (20060101); C12N 9/10 (20060101); C12Q
001/68 (); C12N 001/20 (); C12N 005/00 (); C07H
021/04 () |
Field of
Search: |
;435/193,252.33,410,320.1,6,455 ;536/23.2,23.1,23.6 |
References Cited
[Referenced By]
U.S. Patent Documents
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5073677 |
December 1991 |
Helmer et al. |
5589614 |
December 1996 |
Bridges et al. |
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Foreign Patent Documents
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0 256 223 |
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May 1987 |
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EP |
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WO 93 01294 |
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Jan 1993 |
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WO |
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WO 96/23072 |
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Aug 1996 |
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WO |
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WO 97/11189 |
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Mar 1997 |
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WO |
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WO99/14337 |
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Sep 1999 |
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WO |
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Other References
David C. Holt et al., Characterization of the Safener-Induced
Glutathione S-Transferase Isoform II from Maize, Planta, 196,
295-302, 1995. .
F. Droog, Plant Glutathione S-Transferases, a Tale of Theta and
Tau, J. Plant Growth Regul, 16, 95-107, 1997. .
Laura Rossini et al., Characterization of Glutathione S-Transferase
Isoforms in Three Maise Inbred Lines Exhibiting Differential
Sensitivity to Alachlor, Plant Physiol, 112, 1595-1600, 1996. .
Kathleen A. Marrs, The Functions and Regulation of Glutathione
S-Transferases in Plants, Annu. Rev. Plant Physiol. Plant Mol.
Biol., 47, 127-158, 1996. .
Sharad S. Singhal et al., Purification and Characterization of
Glutathione S-Transferase from Sugarcane Leaves, Phytochemistry,
30, No. 5, 1409-1414, 1991. .
Robert Edwards et al., Glutathione Transferases in Wheat (Triticum)
Species with Activity toward Fenoxaprop-Ethyl and Other Herbicides,
Pesticide Biochemistry and Physiology, 54, 94-104, 1996. .
Michael A. Wosnick et al., Total Chemical Synthesis and Expression
in Escherichia coli of a Maize Glutathione-Transferase (GST) Gene,
Gene, 76, 153-160, 1989. .
Ian Jepson et al., Cloning and Characterization of Maize Herbicide
Safener-induced cDNAs Encoding Subunits of Glutathione
S-Transferase Isoforms I, II, and IV, Plant Molecular Biology, 26,
1855-1866, 1994. .
Dianne A.M. van der Kop et al., Isolation and Characterization of
an Auxin-Inducible Glutathione S-Transferase Gene of Arabidopsis
Thaliana, Plant Molecular Biology, 30, 839-844, 1996. .
Czarnecka et al. Mol. Cell. Biol., 8(3), 1113-1122, 1988. .
Dilip M. Shah et al., Structural Analysis of a Maize Gene Coding
for Glutathione-S-Transferase Involved in Herbicide Detoxification,
Plant Molecular Biology, 6, 203-211, 1986. .
Robert E. Moore et al., Cloning and Expression of a cDNA Encoding a
Maize Glutathione-S-Transferase in E. Coli, Nucleic Acids Research,
14, No. 18, 7227-7235, 1986. .
Kriton K. Hatzios et al., Herbicide Safeners, J. Environ. Sci.
Health, B31(3), 545-553, 1996. .
Thomas Flury et al., A 2,4-D-Inducible Glutathione S-Transferase
from Soybean (Glycine Max)., Physiologia Plantarum, 94, 312-318,
1995. .
Robert Edwards, Characterization of Glutathione Transferases and
Glutathione Peroxidases in Pea, Physiologia Plantarum, 98, 594-604,
1996. .
McGonigle, Brian et al., Hemoglutathione selectivity by soybean,
Pestic. Biochem. Physiol. (1998), 62(1), 15-25. .
Koeppe et al., Role of glutathione conjugation in the
detoxification of sulfonylurea herbicides in plants, Book of
Abstracts, 216.sup.th American Chemical Society, (1998),
(Abstract). .
Grove et al., Characterization and Heterospecific Expression of
CDNA Clones of Genes in the Maize GSH S-Transferase Multigene
Family, Nucleic Acids Research, vol. 16, No. 2, 425-438, Jan. 1,
1988. .
Dixon et al., Purification regulation and cloning of a glutathione
transferase (GST) from maize resembling the auxin-inducible
type-III GST's Plant Molecular Biology, vol. 36, 75-87, Jan., 1998.
.
Wen et al., Expressed sequence tags from B73 maize seedlings, Jun.
8, 1998. .
Nash et al., Bronze-2 gene from maize reconstruction of a wild-type
allele and analysis of transcription and splicing, The Plant Cell,
vol. 2, pp. 1039-1049 1990. .
Marrs et al., A glutathione S-transferase involved in vacuolar
transfer encoded by the maize gene Bronze-2, Nature, vol. 375, Jun.
1, 1995 pp. 397-400. .
Dixon et al., Glutathione-mediated detoxification systems in
plants, Current Opinion in Plant Biology, vol. 1, No. 3, Jun. 1998,
pp. 258-266. .
Timmerman, Molecular Characterization of Corn Glutathione
S-Transferase isozymes involved in Herbicide Detoxification,
Physiologia Plantarum, vol. 77, No. Symp.01, Jan. 1, 1989, pp.
465-471. .
Neuffeind, et al., Plant glutathione S-Transferases and herbicide
detoxification, Biological Chemistry, vol. 378, Mar. 1997 pp.
199-205..
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Primary Examiner: Nashed; Nashaat
Parent Case Text
This is a continuation-in-part of application Ser. No. 08/924,759
filed Sep. 5, 1997, now U.S. Pat. No. 5,962,229.
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding a GST enzyme selected
from the group consisting of:
(a) an isolated nucleic acid fragment encoding the amino acid
sequence selected from the group consisting of SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72 and SEQ ID
NO:74; and
(b) an isolated nucleic acid fragment that is complementary to
(a).
2. The isolated nucleic acid fragment of claim 1 selected from the
group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ
ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID
NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ
ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67,
SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:73.
3. A chimeric gene comprising the isolated nucleic acid fragment of
claim 1 operably linked to suitable regulatory sequences.
4. A transformed host cell comprising the chimeric gene of claim
3.
5. The transformed host cell of claim 4 wherein the host cell is a
plant cell.
6. The transformed host cell of claim 4 wherein the host cell is E.
coli.
7. A method of altering the level of expression of a GST enzyme in
a host cell comprising:
(a) transforming a host cell with the chimeric gene of claim 3
and;
(b) growing the transformed host cell produced in step (a) under
conditions that are suitable for expression of the chimeric gene
resulting in production of altered levels of a GST enzyme in the
transformed host cell relative to expression levels of an
untransformed host cell.
8. A method of obtaining a nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence encoding a GST
enzyme comprising:
(a) probing a cDNA or genomic library with a nucleic acid fragment
selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27,
SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ
ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,
SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEO ID
NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:73,
under the following hybridization conditions: 0.1.times.SSC, 0.1%
SDS at 65 degrees C.;
(b) identifying a DNA clone that hybridizes with the nucleic acid
fragment of step (a); and
(c) sequencing the cDNA or genomic fragment that comprises the
clone identified in step (b),
wherein the sequenced cDNA or genomic fragment encodes all or
substantially all of the amino acid sequence encoding a GST enzyme.
Description
FIELD OF THE INVENTION
This intention is in the field of plant molecular biology. More
specifically, this invention pertains to nucleic acid fragments
encoding maize glutathione-S-transferase (GST) enzymes involved in
the detoxification of xenobiotic compounds in plants and seeds.
BACKGROUND OF THE INVENTION
Glutathione-S-transferases (GST) are a family of enzymes which
catalyze the conjugation of glutathione, homoglutathione (hGSH) and
other glutathione-like analogs via a sulfhydryl group, to a large
range of hydrophobic, electrophilic compounds. The conjugation can
result in detoxification of these compounds. GST enzymes have been
identified in a range of plants including maize (Wosnick et al.,
Gene (Amst) 76 (1) (1989) 153-160; Rossini et al., Plant Physiology
(Rockville) 112 (4) (1996) 1595-1600; Holt et al., Planta
(Heidelberg) 196 (2) (1995) 295-302), wheat (Edwards et al.,
Pestic. Biochem Physiol. (1996) 54(2), 96-104), sorghum (Hatzios et
al., J. Environ. Sci. Health, Part B (1996), B31(3), 545-553),
arabidopsis (Van Der Kop et al., Plant Molecular Biology 30 (4)
(1996), sugarcane (Singhal et al., Phytochemistry (OXF) 30 (5)
(1991) 1409-1414), soybean (Flury et al., Physiologia Plantarum 94
(1995) 594-604) and peas (Edwards R., Physiologia Plantarum 98 (3)
(1996) 594-604). GST's can comprise a significant portion of total
plant protein, for example attaining from 1 to 2% of the total
soluble protein in etiolated maize seedlings (Timmermann, Physiol
Plant. (1989) 77(3), 465-71).
Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the
nucleophilic attack of the thiol group of GSH to various
electrophilic substrates. Their functions and regulation in plants
has been recently reviewed (Marrs et al., Annu Rev Plant Physiol
Plant Mol Biol 47:127-58 (1996); Droog, F. J Plant Growth Regul
16:95-107, (1997)). They are present at every stage of plant
development from early embryogenesis to senescence and in every
tissue class examined. The agents that have been shown to cause an
increase in GST levels have the potential to cause oxidative
destruction in plants, suggesting a role for GSTs in the protection
from oxidative damage. In addition to their role in the protection
from oxidative damage, GSTs have the ability to nonenzymatically
bind certain small molecules, such as auxin (Zettl et al., PNAS
91:689-693, (1994)) and perhaps regulate their bioavailability.
Furthermore the addition of GSH to a molecule serves as an
"address" to send that molecule to the plant vacuole (Marrs et al.,
Nature 375:397-400, (1995)).
GSTs have also been implicated in the detoxification of certain
herbicides. Maize GSTs have been well characterized in relation to
herbicide metabolism. Three genes from maize have been cloned: GST
29 (Shah et al., Plant Mol Biol 6, 203-211(1986)), GST 27 (Jepson
et al., Plant Mol Biol 26:1855-1866, (1994)), GST 26 (Moore et al.,
Nucleic Acids Res 14:7227-7235 (1986)). These gene products form
four GST isoforms: GST I (a homodimer of GST 29), GST II (a
heterodimer of GST 29 and GST 27), GST III (a homodimer of GST 26),
and GST IV (a homodimer of GST 27). GST 27 is
highly inducible by safener compounds (Jepson (1994) supra; Holt et
al., Planta 196:295-302, (1995)) and overexpression of GST 27 in
tobacco confers alachlor resistance to transgenic tobacco (Jepson,
personal communication). Additionally, Bridges et al. (U.S. Pat.
No. 5,589,614) disclose the sequence of a maize derived GST isoform
II promoter useful for the expression of foreign genes in maize and
wheat. In soybean, herbicide compounds conjugated to hGSH have been
detected and correlated with herbicide selectivity (Frear et al.,
Physiol 20: 299-310 (1983); Brown et al., Pest Biochem Physiol
29:112-120, (1987)). This implies that hGSH conjugation is an
important determinant in soybean herbicide selectivity although
this hypothesis has not been characterized on a molecular
level.
Some efforts have been made to alter plant phenotypes by the
expression of either plant or mammalian foreign GST genes or their
promoters in mature plant tissue. For example, Helmer et al. (U.S.
Pat No. 5,073,677) teach the expression of a rat GST gene in
tobacco under the control of a strong plant promoter. Similarly,
Jepson et al. (WO 97/11189) disclose a chemically inducible maize
GST promoter useful for the expression of foreign proteins in
plants; Chilton et al. (EP 256223) discuss the construction of
herbicide tolerant plants expressing a foreign plant GST gene; and
Bieseler et al. (WO 96/23072) teach DNA encoding GSTIIIc, its
recombinant production and transgenic plants containing the DNA
having a herbicide-tolerant phenotype.
Manipulation of nucleic acid fragments encoding soybean GST to use
in screening in assays, the creation of herbicide-tolerant
transgenic plants, and altered production of GST enzymes depend on
the heretofore unrealized isolation of nucleic acid fragments that
encode all or a substantial portion of a soybean GST enzyme.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid fragments isolated from
maize encoding all or a substantial portion of a GST enzyme. The
isolated nucleic acid fragment is selected from the group
consisting of (a) an isolated nucleic acid fragment encoding all or
a substantial portion of the amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ
ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ
ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,
SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72 and
SEQ ID NO:74; (b) an isolated nucleic acid fragment that is
substantially similar to an isolated nucleic acid fragment encoding
all or a substantial portion of the amino acid sequence sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,
SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID
NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ
ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,
SEQ ID NO:72 and SEQ ID NO:74.; and (c) an isolated nucleic acid
fragment that is complementary to (a) or (b). The nucleic acid
fragments and corresponding polypeptides are contained in the
accompanying Sequence Listing and described in the Brief
Description of the Invention.
In another embodiment, the instant invention relates to chimeric
genes encoding maize GST enzymes or to chimeric genes that comprise
nucleic acid fragments as described above, the chimeric genes
operably linked to suitable regulatory sequences, wherein
expression of the chimeric genes results in altered levels of the
encoded enzymes in transformed host cells.
The present invention further provides a transformed host cell
comprising the above described chimeric gene. The transformed host
cells can be of eukaryotic or prokaryotic origin. The invention
also includes transformed plants that arise from transformed host
cells of higher plants, and from seeds derived from such
transformed plants, and subsequent progeny.
Additionally, the invention provides methods of altering the level
of expression of a maize GST enzyme in a host cell comprising the
steps of; (i) transforming a host cell with the above described
chimeric gene and; (ii) growing the transformed host cell produced
in step (i) under conditions that are suitable for expression of
the chimeric gene wherein expression of the chimeric gene results
in production of altered levels of a plant GST enzyme in the
transformed host cell relative to expression levels of an
untransformed host cell.
In an alternate embodiment, the present invention provides methods
of obtaining a nucleic acid fragment encoding all or substantially
all of the amino acid sequence encoding a maize GST enzyme
comprising either hybridization or primer-directed amplification
methods known in the art and using the above described nucleic acid
fragment. A primer-amplification-based method uses SEQ ID NOS.:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71
or 73. The product of these methods is also part of the
invention.
Another embodiment of the invention includes a method for
identifying a compound that inhibits the activity of a maize GST
enzyme encoded by the nucleic acid fragment and substantially
similar and complementary nucleic acid fragments of SEQ ID NOS.:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71
and 73. The method has the steps: (a) transforming a host cell with
the above described chimeric gene; (b) growing the transformed host
cell under conditions that are suitable for expression of the
chimeric gene wherein expression of the chimeric gene results in
production of the GST enzyme; (c) optionally purifying the GST
enzyme expressed by the transformed host cell; (d) contacting the
GST enzyme with a chemical compound of interest; and (e)
identifying the chemical compound of interest that reduces the
activity of the maize GST enzyme relative to the activity of the
maize GST enzyme in the absence of the chemical compound of
interest.
This method may further include conducting step (d) in the presence
of at least one electrophilic substrate and at least one thiol
donor. The isolated nucleic acid fragments of this method are
chosen from the group represented by SEQ ID NOS.:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73 and the
maize GST enzyme is selected from the group consisting of SEQ ID
NOS.:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, and 74.
The invention further provides a method for identifying a chemical
compound that inhibits the activity of the maize GST enzyme as
described herein, wherein the identification is based on a
comparison of the phenotype of a plant transformed with the above
described chimeric gene contacted with the inhibitor candidate with
the phenotype of a transformed plant that is not contacted with the
inhibitor candidate. The isolated nucleic acid fragment of this
method is selected from the group consisting of SEQ ID NOS.:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71
and 73 and the maize GST enzyme is selected from the group
consisting of SEQ ID NOS.:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68, 70, 72, and 74.
In another embodiment, the invention provides a method for
identifying a substrate for the maize GST enzyme. The method
comprises the steps of: (a) transforming a host cell with a
chimeric gene comprising the nucleic acid fragment as described
herein, the chimeric gene encoding a maize GST enzyme operably
linked to at least one suitable regulatory sequence; (b) growing
the transformed host cell of step (a) under conditions that are
suitable for expression of the chimeric gene resulting in
production of the GST enzyme; (c) optionally purifying the GST
enzyme expressed by the transformed host cell; (d) contacting the
GST enzyme with a substrate candidate; and (e) comparing the
activity of maize GST enzyme with the activity of maize GST enzyme
that has been contacted with the substrate candidate and selecting
substrate candidates that increase the activity of the maize GST
enzyme relative to the activity of maize GST enzyme in the absence
of the substrate candidate. More preferably, step (d) of this
method is carried out in the presence of at least one thiol donor.
The isolated nucleic acid fragment of this method is selected from
the group consisting of SEQ ID NOS.:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73 and the maize GST
enzyme is selected from the group consisting of SEQ ID NOS.:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
and 74.
Alternatively, methods are provided for identifying a maize GST
substrate candidate wherein the identification of the substrate
candidate is based on a comparison of the phenotype of a host cell
transformed with a chimeric gene expressing a maize GST enzyme and
contacted with a substrate candidate with the phenotype of a
similarly transformed host cell grown without contact with a
substrate candidate.
The isolated nucleic acid fragment of this method is selected from
the group consisting of SEQ ID NOS.:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73 and the maize GST
enzyme is selected from the group consisting of SEQ ID NOS.:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
and 74.
BRIEF DESCRIPTION OF SEQUENCE DESCRIPTIONS AND BIOLOGICAL
DEPOSITS
The invention can be more fully understood from the following
detailed description and the accompanying sequence descriptions and
biological deposits which form a part of this application.
The following sequence descriptions and sequences listings attached
hereto comply with the rules governing nucleotide and/or amino acid
sequence disclosures in patent applications as set forth in 37
C.F.R. .sctn.1.821-1.825. The Sequence Descriptions contain the one
letter code for nucleotide sequence characters and the three letter
codes for amino acids as defined in conformity with the IUPAC-IYUB
standards described in Nucleic Acids Research 13:3021-3030 (1985)
and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are
herein incorporated by reference.
SEQ ID NO:1 is the nucleotide sequence comprising the cDNA insert
in clone bms1.pk0023.g8 encoding a maize GST.
SEQ ID NO:2 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone bms1.pk0023.g8.
SEQ ID NO:3 is the nucleotide sequence comprising the cDNA insert
in clone cs.pk0010.c5 encoding a maize GST.
SEQ ID NO:4 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cs.pk0010.c5.
SEQ ID NO:5 is the nucleotide sequence comprising the cDNA insert
in clone ceb1.pk0017.a5 encoding a maize GST.
SEQ ID NO:6 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ceb1.pk0017.a5.
SEQ ID NO:7 is the nucleotide sequence comprising the cDNA insert
in clone cc71se-a.pk0001.g2 encoding a maize class III GST.
SEQ ID NO:8 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone
cc71se-a.pk0001.g2.
SEQ ID NO:9 is the nucleotide sequence comprising the cDNA insert
in clone cc71se-b.pk0014.b8 encoding a maize class III GST.
SEQ ID NO:10 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone
cc71se-b.pk0014.b8.
SEQ ID NO:11 is the nucleotide sequence comprising the cDNA insert
in clone ceb5.pk0051.f8 encoding a maize class III GST.
SEQ ID NO:12 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ceb5.pk0051.f8.
SEQ ID NO:13 is the nucleotide sequence comprising the cDNA insert
in clone cr1n.pk0003.b1 encoding a maize class III GST.
SEQ ID NO:14 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1n.pk0003.b1.
SEQ ID NO:15 is the nucleotide sequence comprising the cDNA insert
in clone cr1n.pk0014.g8 encoding a maize class III GST.
SEQ ID NO:16 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1n.pk0014.g8.
SEQ ID NO:17 is the nucleotide sequence comprising the cDNA insert
in clone m.15.5.d06.sk20 encoding a maize class II GST.
SEQ ID NO:18 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone m.15.5.d06.sk20.
SEQ ID NO:19 is the nucleotide sequence comprising the cDNA insert
in clone cr1n.pk0040.e12 encoding a maize class II GST.
SEQ ID NO:20 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1n.pk0040.e12.
SEQ ID NO:21 is the nucleotide sequence comprising the cDNA insert
in clone ceb5.pk0049.a11 encoding a maize class III GST.
SEQ ID NO:22 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ceb5.pk0049.a11.
SEQ ID NO:23 is the nucleotide sequence comprising the cDNA insert
in clone cs1.pk0059.e2 encoding a maize class III GST.
SEQ ID NO:24 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cs1.pk0059.e2.
SEQ ID NO:25 is the nucleotide sequence comprising the cDNA insert
in clone cbn2.pk0032.d10 encoding a maize class I GST.
SEQ ID NO:26 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cbn2.pk0032.d10
SEQ ID NO:27 is the nucleotide sequence comprising the cDNA insert
in clone cr1n.pk0164.g7 encoding a maize class I GST.
SEQ ID NO:28 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1n.pk0164.g7
SEQ ID NO:29 is the nucleotide sequence comprising the cDNA insert
in clone cdt2c.pk003.115 encoding a maize class I GST.
SEQ ID NO:30 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cdt2c.pk003.115
SEQ ID NO:31 is the nucleotide sequence comprising the cDNA insert
in clone csc1c.pk001.h7 encoding a maize class I GST.
SEQ ID NO:32 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone csc1c.pk001.h7
SEQ ID NO:31 is the nucleotide sequence comprising the cDNA insert
in clone csc1c.pk001.h7 encoding a maize class I GST.
SEQ ID NO:32 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone csc1c.pk001.h7
SEQ ID NO: 33 is the nucleotide sequence comprising the cDNA insert
in clone p0110.cgsnt78r encoding a maize class I GST.
SEQ ID NO:34 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0110.cgsnt78r.
SEQ ID NO:35 is the nucleotide sequence comprising the cDNA insert
in clone p0121.cfrmz42r encoding a maize class I GST.
SEQ ID NO:36 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0121.cfrmz42r.
SEQ ID NO:37 is the nucleotide sequence comprising the cDNA insert
in clone csi1n.pk0034.a11 encoding a maize class III GST.
SEQ ID NO:38 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone csi1n.pk0034.a11.
SEQ ID NO:39 is the nucleotide sequence comprising the cDNA insert
in clone cepe7.pk0028.g3 encoding a maize class III GST.
SEQ ID NO:40 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cepe7.pk0028.g3.
SEQ ID NO:41 is the nucleotide sequence comprising the cDNA insert
in clone cr1n.pk0167.d7 encoding a maize class III GST.
SEQ ID NO:42 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1n.pk0167.d7.
SEQ ID NO:43 is the nucleotide sequence comprising the cDNA insert
in clone cco1.pk0027.e4 encoding a maize class III GST.
SEQ ID NO:44 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cco1.pk0027.e4.
SEQ ID NO:45 is the nucleotide sequence comprising the cDNA insert
in clone cpj1c.pk001.d21 encoding a maize class III GST.
SEQ ID NO:46 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cpj1c.pk001.d21.
SEQ ID NO:47 is the nucleotide sequence comprising the cDNA insert
in clone cse1c.pk001.b8 encoding a maize class III GST.
SEQ ID NO:48 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cse1c.pk001.b8.
SEQ ID NO:49 is the nucleotide sequence comprising the cDNA insert
in clone cr1s.pk010.f1 encoding a maize class III GST.
SEQ ID NO:50 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cr1s.pk010.f1.
SEQ ID NO:51 is the nucleotide sequence comprising the cDNA insert
in clone cpf1c.pk002.a13 encoding a maize class III GST.
SEQ ID NO:52 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cpf1c.pk002.a13.
SEQ ID NO:53 is the nucleotide sequence comprising the cDNA insert
in clone cho1c.pk004.c15 encoding a maize class III GST.
SEQ ID NO:54 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cho1c.pk004.c15.
SEQ ID NO:55 is the nucleotide sequence comprising the cDNA insert
in clone cpi1c.pk002.m4 encoding a maize class III GST.
SEQ ID NO:56 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone cpi1c.pk002.m4.
SEQ ID NO:57 is the nucleotide sequence comprising the cDNA insert
in clone chpc8.pk057.f10 encoding a maize class III GST.
SEQ ID NO:58 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone chpc8.pk057.f10.
SEQ ID NO:59 is the nucleotide sequence comprising the cDNA insert
in clone p0014.ctu90r encoding a maize class III GST.
SEQ ID NO:60 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0014.ctu90r.
SEQ ID NO:61 is the nucleotide sequence comprising the cDNA insert
in p0006.cbyvs55r encoding a maize class III GST.
SEQ ID NO:62 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0006.cbyvs55r.
SEQ ID NO:63 is the nucleotide sequence comprising the cDNA insert
in p0037.crwaf68r encoding a maize class III GST.
SEQ ID NO:64 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0037.crwaf68r.
SEQ ID NO:65 is the nucleotide sequence comprising the cDNA insert
in p0032.crcas61r encoding a maize class III GST.
SEQ ID NO:66 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0032.crcas61r.
SEQ ID NO:67 is the nucleotide sequence comprising the cDNA insert
in p0088.clrim45r encoding a maize class III GST.
SEQ ID NO:68 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0088.clrim45r.
SEQ ID NO:69 is the nucleotide sequence comprising the cDNA insert
in p0126.cnlag50r encoding a maize class III GST.
SEQ ID NO:70 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0126.cnlag50r.
SEQ ID NO:71 is the nucleotide sequence comprising the cDNA insert
in p0095.cwsba73r encoding a maize class III GST.
SEQ ID NO:72 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0095.cwsba73r.
SEQ ID NO:73 is the nucleotide sequence comprising the cDNA insert
in p0125.czaaj03r encoding a maize class III GST.
SEQ ID NO:74 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone p0125.czaaj03r.
The transformed E. coli ceb5.pk0051.f8/pET30(LIC)BL21(DE3)
containing the gene ceb5.pk0051.f8 in a pET30(LIC) vector encoding
a maize class III GST was deposited on Aug. 21, 1997 with the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, U.S.A., under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Micro-organisms for the Purpose of Patent Procedure. The deposit is
designated as ATCC 98511.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel GST nucleotide sequences and
encoded proteins isolated from maize. GST enzymes are known to
function in the process of detoxification of a variety of
xenobiotic compounds in plants, most notably, herbicides. Nucleic
acid fragments encoding at least a portion of several maize GST
enzymes have been isolated and identified by comparison of random
plant cDNA sequences to public databases containing nucleotide and
protein sequences using the BLAST algorithms well known to those
skilled in the art. The sequences of the present invention are
useful in the construction of herbicide-tolerant transgenic plants,
in the recombinant production of GST enzymes, in the development of
screening assays to identify compounds inhibitory to the GST
enzymes, and in screening assays to identify chemical substrates of
the GSTs.
In the context of this disclosure, a number of terms shall be
utilized.
As use herein "Glutathione S-Transferase" or "GST" refers to any
plant derived glutathione S-transferase (GST) enzyme capable of
catalyzing the conjugation of glutathione, homoglutathione and
other glutathione-like analogs via a sulfhydryl group, to
hydrophobic and electrophilic compounds. The term GST includes
amino acid sequences longer or shorter than the length of natural
GSTs, such as functional hybrid or partial fragments of GSTs, or
their analogues. As used herein "GST" is not intended to be
delimited on the basis of enzyme activity but may encompass amino
acid sequences that possess no measurable enzyme activity but are
substantially similar in to those sequences, known in the art to
possess the above mentioned glutathione conjugating activity.
The term "class" or "GST class" refers to a grouping of the various
GST enzymes according to amino acid identity. Currently, four
classes have been identified and are referred to as "GST class I"
"GST class II", "GST class III" and "GST class IV". The grouping of
plant GSTs into three classes is described by Droog et al. (Plant
Physiology 107:1139-1146 (1995)). All available amino acid
sequences were aligned using the Wisconsin Genetics Computer Group
package (Wisconsin Package Version 9.0, Genetics Computer Group
(GCG), Madison, Wis.), and graphically represented on a
phylogenetic tree. Three groups were identified: class one
including the archetypical sequences from maize GST I (X06755) and
GST III (X04375); class two including the archetypical sequence
from Dianthus caryophyllus (M64628); and class three including the
archetypical sequence soybean GH2/4 (M20363). Recently, Applicants
have established a further subgroup of the plant GSTs known as
class IV GSTs with its archetypical sequence being In2-1
(X58573).
As used herein, an "isolated nucleic acid fragment" is a polymer of
RNA or DNA that is single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases. An
isolated nucleic acid fragment in the form of a polymer of DNA may
be comprised of one or more segments of cDNA, genomic DNA or
synthetic DNA.
As used herein, "substantially similar" refers to nucleic acid
fragments wherein changes in one or more nucleotide bases results
in substitution of one or more amino acids, but do not affect the
functional properties of the protein encoded by the DNA sequence.
"Substantially similar" also refers to nucleic acid fragments
wherein changes in one or more nucleotide bases does not affect the
ability of the nucleic acid fragment to mediate alteration of gene
expression by antisense or co-suppression technology.
"Substantially similar" also refers to modifications of the nucleic
acid fragments of the instant invention such as deletion or
insertion of one or more nucleotide bases that do not substantially
affect the functional properties of the resulting transcript
vis-a-vis the ability to mediate alteration of gene expression by
antisense or co-suppression technology or alteration of the
functional properties of the resulting protein molecule. It is
therefore understood that the invention encompasses more than the
specific exemplary sequences.
For example, it is well known in the art that antisense suppression
and co-suppression of gene expression may be accomplished using
nucleic acid fragments representing less that the entire coding
region of a gene, and by nucleic acid fragments that do not share
100% identity with the gene to be suppressed. Moreover, alterations
in a gene which result in the production of a chemically equivalent
amino acid at a given site, but do not effect the functional
properties of the encoded protein, are well known in the art. Thus,
a codon for the amino acid alanine, a hydrophobic amino acid, may
be substituted by a codon encoding another less hydrophobic residue
(such as glycine) or a more hydrophobic residue (such as valine,
leucine, or isoleucine). Similarly, changes which result in
substitution of one negatively charged residue for another (such as
aspartic acid for glutamic acid) or one positively charged residue
for another (such as lysine for arginine) can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the protein molecule would also not be expected to alter the
activity of the protein. Each of the proposed modifications is well
within the routine skill in the art, as is determination of
retention of biological activity of the encoded products. Moreover,
the skilled artisan recognizes that substantially similar sequences
encompassed by this invention are also defined by their ability to
hybridize, under stringent conditions (0.1.times.SSC, 0.1% SDS,
65.degree. C.), with the sequences exemplified herein. Preferred
substantially similar nucleic acid fragments of the instant
invention are those nucleic acid fragments whose DNA sequences are
at least 80% identical to the DNA sequence of the nucleic acid
fragments reported herein. More preferred nucleic acid fragments
are at least 95% identical to the DNA sequence of the nucleic acid
fragments reported herein.
A nucleic acid molecule is "hybridizable" to another nucleic acid
molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor (1989), particularly Chapter 11 and Table 11.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. For preliminary screening for homologous nucleic
acids, low stringency hybridization conditions, corresponding to a
Tm of 55.sup.o, can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS.
Moderate stringency hybridization conditions correspond to a higher
Tm, e.g., 40% formamide, with 5.times. or 6.times.SSC.
Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferable a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least 30 nucleotides. Furthermore,
the skilled artisan will recognize that the temperature and wash
solution salt concentration may be adjusted as necessary according
to factors such as length of the probe.
A "substantial portion" of an amino acid or nucleotide sequence
comprising enough of the amino acid sequence of a polypeptide or
the nucleotide sequence of a gene to putatively identify that
polypeptide or gene, either by manual evaluation of the sequence by
one skilled in the art, or by computer-automated sequence
comparison and identification using algorithms such as BLAST (Basic
Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol
Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In
general, a sequence of ten or more contiguous amino acids or thirty
or more nucleotides is necessary in order to putatively identify a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene specific oligonucleotide probes comprising 20-30 contiguous
nucleotides may be used in sequence-dependent methods of gene
identification (e.g., Southern hybridization) and isolation (e.g.,
in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12-15 bases may be
used as amplification primers in PCR in order to obtain a
particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial protion" of a nucleotide sequence
comprises enough of the sequence to specifically identify and/or
isolate a nucleic acid fragment comprising the sequence. The
instant specification teaches partial or complete amino acid and
nucleotide sequences encoding one or more particular fungal
proteins. The skilled artisan, having the benefit of the sequences
as reported herein, may now use all or a substantial portion of the
disclosed sequences for purposes known to those skilled in this
art. Accordingly, the instant invention comprises the complete
sequences as reported in the accompanying Sequence Listing, as well
as substantial portions of those sequences as defined above.
The term "percent identity", as known in the art, is a relationship
between two or more polypeptide sequences or two or more
polynucleotide sequences, as determined by comparing the sequences.
In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991). Preferred methods to
determine identity are designed to give the largest match between
the sequences tested. Methods to determine identity and similarity
are codified in publicly available computer programs. Preferred
computer program methods to determine identity and similarity
between two sequences include, but are not limited to, the GCG
Pileup program found in the GCG program package, as used in the
instant invention, using the Needleman and Wunsch algorithm with
their standard default values of gap creation penalty=12 and gap
extension penalty=4 (Devereux et al., Nucleic Acids Res. 12:387-395
(1984)), BLASTP, BLASTN,
and FASTA (Pearson et al., Proc. Natl. Acad Sci. U.S.A.
85:2444-2448 (1988). The BLAST X program is publicly available from
NCBI and other sources (BLAST Manual, Altschul et al., Natl. Cent.
Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md.
20894; Altschul et al., J. Mol. Biol 215:403-410 (1990)). Another
preferred method to determine percent identity, is by the method of
DNASTAR protein alignment protocol using the Jotun-Hein algorithm
(Hein et al., Methods Enzymol. 183:626-645 (1990)). Default
parameters for the Jotun-Hein method for alignments are: for
multiple alignments, gap penalty=11, gap length penalty=3; for
pairwise alignments ktuple=6. As an illustration, by a
polynucleotide having a nucleotide sequence having at least, for
example, 95% "identity" to a reference nucleotide sequence of SEQ
ID NO:1 it is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that
the polynucleotide sequence may include up to five point mutations
per each 100 nucleotides of the reference nucleotide sequence of
SEQ ID NO:1. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. Analogously, by a
polypeptide having an amino acid sequence having at least, for
example, 95% identity to a reference amino acid sequence of SEQ ID
NO:2 is intended that the amino acid sequence of the polypeptide is
identical to the reference sequence except that the polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the reference amino acid of SEQ ID NO:2. In other
words, to obtain a polypeptide having an amino acid sequence at
least 95% identical to a reference amino acid sequence, up to 5% of
the amino acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up
to 5% of the total amino acid residues in the reference sequence
may be inserted into the reference sequence. These alterations of
the reference sequence may occur at the amino or carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
The term "complementary" is used to describe the relationship
between nucleotide bases that are capable to hybridizing to one
another. For example, with respect to DNA, adenosine is
complementary to thymine and cytosine is complementary to guanine.
Accordingly, the instant invention also includes isolated nucleic
acid fragments that are complementary to the complete sequences as
reported in the accompanying Sequence Listing as well as those
substantially similar nucleic acid sequences.
"Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment that encodes
all or a substantial portion of the amino acid sequence encoding
the GST enzymes as set forth in SEQ ID Nos: SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:72 and SEQ ID NO:74. The skilled artisan is well
aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a gene for improved expression in a
host cell, it is desirable to design the gene such that its
frequency of codon usage approaches the frequency of preferred
codon usage of the host cell.
"Synthetic genes" can be assembled from oligonucleotide building
blocks that are chemically synthesized using procedures known to
those skilled in the art These building blocks are ligated and
annealed to form gene segments which are then enzymatically
assembled to construct the entire gene. "Chemically synthesized",
as related to a sequence of DNA, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
DNA may be accomplished using well established procedures, or
automated chemical synthesis can be performed using one of a number
of commercially available machines. Accordingly, the genes can be
tailored for optimal gene expression based on optimization of
nucleotide sequence to reflect the codon bias of the host cell. The
skilled artisan appreciates the likelihood of successful gene
expression if codon usage is biased towards those codons favored by
the host. Determination of preferred codons can be based on a
survey of genes derived from the host cell where sequence
information is available.
"Gene" refers to a nucleic acid fragment that expresses a specific
protein, including regulatory sequences preceding (5' non-coding
sequences) and following (3' non-coding sequences) the coding
sequence. "Native gene" refers to a gene as found in nature with
its own regulatory sequences. "Chimeric gene" refers any gene that
is not a native gene, comprising regulatory and coding sequences
that are not found together in nature. Accordingly, a chimeric gene
may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of an organism. A
"foreign" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure.
"Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Suitable regulatory sequences" refer
to nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
"Promoter" refers to a DNA sequence capable of controlling the
expression of a coding sequence or functional RNA. In general, a
coding sequence is located 3' to a promoter sequence. The promoter
sequence consists of proximal and more distal upstream elements,
the latter elements often referred to as enhancers. Accordingly, an
"enhancer" is a DNA sequence which can stimulate promoter activity
and may be an innate element of the promoter or a heterologous
element inserted to enhance the level or tissue-specificity of a
promoter. Promoters may be derived in their entirety from a native
gene, or be composed of different elements derived from different
promoters found in nature, or even comprise synthetic DNA segments.
It is understood by those skilled in the art that different
promoters may direct the expression of a gene in different tissues
or cell types, or at different stages of development, or in
response to different environmental conditions. Promoters which
cause a gene to be expressed in most cell types at most times are
commonly referred to as "constitutive promoters". New promoters of
various types useful in plant cells are constantly being
discovered; numerous examples may be found in the compilation by
Okamuro and Goldberg, (1989) Biochemistry of Plants 15:1-82. It is
further recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, DNA
fragments of different lengths may have identical promoter
activity.
The "translation leader sequence" refers to a DNA sequence located
between the promoter sequence of a gene and the coding sequence.
The translation leader sequence is present in the fully processed
mRNA upstream of the translation start sequence. The translation
leader sequence may affect processing of the primary transcript to
mRNA, mRNA stability or translation efficiency. Examples of
translation leader sequences have been described (Turner, R. and
Foster, G. D. (1995) Molecular Biotechnology 3:225).
The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. ((1989) Plant Cell
1:671-680).
"RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene (U.S. Pat. No. 5,107,065). The complementarity of an
antisense RNA may be with any part of the specific gene transcript,
i.e., at the 5' non-coding sequence, 3' non-coding sequence,
introns, or the coding sequence. "Functional RNA" refers to
antisense RNA, ribozyme RNA, or other RNA that is not translated
yet has an effect on cellular processes.
The term "operably linked" refers to the association of nucleic
acid sequences on a single nucleic acid fragment so that the
function of one is affected by the other. For example, a promoter
is operably linked with a coding sequence when it is capable of
affecting the expression of that coding sequence (i.e., that the
coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in sense or antisense orientation.
The term "expression", as used herein, refers to the transcription
and stable accumulation of sense (mRNA) or antisense RNA derived
from the nucleic acid fragment of the invention. Expression may
also refer to translation of mRNA into a polypeptide. "Antisense
inhibition" refers to the production of antisense RNA transcripts
capable of suppressing the expression of the target protein.
"Overexpression" refers to the production of a gene product in
transgenic organisms that exceeds levels of production in normal or
non-transformed organisms. "Co-suppression" refers to the
production of sense RNA transcripts capable of suppressing the
expression of identical or substantially similar foreign or
endogenous genes (U.S. Pat. No. 5,231,020).
"Altered levels" refers to the production of gene product(s) in
transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
"Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
A "chloroplast transit peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the chloroplast or other plastid types present in the cell in which
the protein is made. "Chloroplast transit sequence" refers to a
nucleotide sequence that encodes a chloroplast transit peptide. A
"signal peptide" is an amino acid sequence which is translated in
conjunction with a protein and directs the protein to the secretory
system (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol.
Biol. 42:21-53). If the protein is to be directed to a vacuole, a
vacuolar targeting signal (supra) can further be added, or if to
the endoplasmic reticulum, an endoplasmic reticulum retention
signal (supra) may be added. If the protein is to be directed to
the nucleus, any signal peptide present should be removed and
instead a nuclear localization signal included (Raikhel (1992)
Plant Phys.100:1627-1632).
"Transformation" refers to the transfer of a nucleic acid fragment
into the genome of a host organism, resulting in genetically stable
inheritance. Host organisms containing the transformed nucleic acid
fragments are referred to as "transgenic" organisms. Examples of
methods of plant transformation include Agrobacterium-mediated
transformation (De Blaere et al. (1987) Meth Enzymol. 143:277) and
particle-accelerated or "gene gun" transformation technology (Klein
et al. (1987) Nature (London) 327:70-73; U.S. Pat. No.
4,945,050).
The term "herbicide-tolerant plant" as used herein is defined as a
plant that survives and preferably grows normally at a usually
effective dose of a herbicide. Herbicide tolerance in plants
according to the present invention refers to detoxification
mechanisms in a plant, although the herbicide binding or target
site is still sensitive.
"Thiol donor" refers to a compound that contains the structure RSH
(where R is not equal to H). Within the context of the present
invention suitable thiol donors may include, but are not limited
to, Glutathione and homoglutathione.
"Electrophilic substrate" refers to a compound that is amenable to
conjugation with glutathione or homoglutathione via a sulfhydryl
group. Electrophilic substrates include a wide variety of compounds
including pesticides, anti-pathogenic compounds such as fungicides
and profungicides, pheramones, and herbicides. Within the context
of the present invention electrophilic substrates with herbicidal
activity may include, but are not limited to, chlorimuronethyl,
alachlor, and atrazine, 1-chloro-2,4-dinitrobenzene (CDNB),
ethacrynic acid, t-stilbene oxide, and
1,2-epoxy-3-(p-nitrophenoxy)propane.
Standard recombinant DNA and molecular cloning techniques used
herein are well known in the art and are described more fully in
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, 1989 (hereinafter "Maniatis").
"Thiol donor" refers to a compound that contains the structure RSH
(where R is not equal to H). Within the context of the present
invention suitable thiol donors may include, but are not limited
to, Glutathione and homoglutathione.
"Electrophilic substrate" refers to a compound that is amenable to
conjugation with glutathione or homoglutathione via a sulfhydryl
group. Electrophilic substrates include a wide variety of compounds
including pesticides, anti-pathogenic compounds such as fungicides
and profungicides, pheramones, and herbicides. Within the context
of the present invention electrophilic substrates with herbicidal
activity may include, but are not limited to, chlorimuronethyl,
alachlor, and atrazine, 1-chloro-2,4-dinitrobenzene (CDNB),
ethacrynic acid, t-stilbene oxide, and
1,2-epoxy-3-(p-nitrophenoxy)propane.
The nucleic acid fragments of the instant invention may be used to
isolate cDNAs and genes encoding homologous enzymes from the same
or other plant species. Isolation of homologous genes using
sequence-dependent protocols is well known in the art. Examples of
sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase
chain reaction).
For example, genes encoding other GST enzymes, either as cDNAs or
genomic DNAs, could be isolated directly by using all or a portion
of the instant nucleic acid fragments as DNA hybridization probes
to screen libraries from any desired plant using methodology well
known to those skilled in the art. Specific oligonucleotide probes
based upon the instant nucleic acid sequences can be designed and
synthesized by methods known in the art (Maniatis). Moreover, the
entire sequences can be used directly to synthesize DNA probes by
methods known to the skilled artisan such as random primers DNA
labeling, nick translation, or end-labeling techniques, or RNA
probes using available in vitro transcription systems. In addition,
specific primers can be designed and used to amplify a part of or
full-length of the instant sequences. The resulting amplification
products can be labeled directly during amplification reactions or
labeled after amplification reactions, and used as probes to
isolate full length cDNA or genomic fragments under conditions of
appropriate stringency.
In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al., (1988) PNAS USA 85:8998) to generate
cDNAs by using PCR to amplify copies of the region between a single
point in the transcript and the 3' or 5' end. Primers oriented in
the 3' and 5' directions can be designed from the instant
sequences. Using commercially available 3' RACE or 5' RACE systems
(BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et
al., (1989) PNAS USA 86:5673; Loh et al., (1989) Science 243:217).
Products generated by the 3' and 5' RACE procedures can be combined
to generate full-length cDNAs (Frohman, M. A. and Martin, G. R.,
(1989) Techniques 1:165).
Availability of the instant nucleotide and deduced amino acid
sequences facilitates immunological screening cDNA expression
libraries. Synthetic peptides representing portions of the instant
amino acid sequences may be synthesized. These peptides can be used
to immunize animals to produce polyclonal or monoclonal antibodies
with specificity for peptides or proteins comprising the amino acid
sequences. These antibodies can be then be used to screen cDNA
expression libraries to isolate full-length cDNA clones of interest
(Lerner, R. A. (1984) Adv. Immunol. 36:1; Maniatis).
The nucleic acid fragments of the instant invention may be used to
create transgenic plants in which the disclosed GST enzymes are
present at higher or lower levels than normal or in cell types or
developmental stages in which they are not normally found. This
would have the effect of altering the level of GST enzyme available
as well as the herbicide tolerant-phenotype of the plant.
Overexpression of the GST enzymes of the instant invention may be
accomplished by first constructing chimeric genes in which the
coding region are operably linked to promoters capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric genes may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals must also be provided. The
instant chimeric genes may also comprise one or more introns in
order to facilitate gene expression.
Any combination of any promoter and any terminator capable of
inducing expression of a GST coding region may be used in the
chimeric genetic sequence. Some suitable examples of promoters and
terminators include those from nopaline synthase (nos), octopine
synthase (ocs) and cauliflower mosaic virus (CaMV) genes. One type
of efficient plant promoter that may be used is a high level plant
promoter. Such promoters, in operable linkage with the genetic
sequence for GST, should be capable of promoting expression of the
GST such that the transformed plant is tolerant to an herbicide due
to the presence of, or increased levels of, GST enzymatic activity.
High level plant promoters that may be used in this invention
include the promoter of the small subunit (ss) of the
ribulose-1,5-bisphosphate carboxylase from example from soybean
(Berry-Lowe et al., J. Molecular and App. Gen., 1:483-498 1982)),
and the promoter of the chlorophyll a/b binding protein. These two
promoters are known to be light-induced in plant cells (See, for
example, Genetic Engineering of Plants, an Agricultural
Perspective, A. Cashmore, Plenum, New York (1983), pages 29-38;
Coruzzi, G. et al., The Journal of Biological Chemistry, 258:1399
(1983), and Dunsmuir, P. et al., Journal of Molecular and Applied
Genetics, 2:285 (1983)).
Plasmid vectors comprising the instant chimeric genes can then
constructed. The choice of plasmid vector depends upon the method
that will be used to transform host plants. The skilled artisan is
well aware of the genetic elements that must be present on the
plasmid vector in order to successfully transform, select and
propagate host cells containing the chimeric gene. The skilled
artisan will also recognize that different independent
transformation events will result in different levels and patterns
of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida
et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that
multiple events must be screened in order to obtain lines
displaying the desired expression level and pattern. Such screening
may be accomplished by Southern analysis of DNA blots (Southern, J.
Mol. Biol. 98, 503, (1975)). Northern analysis of mRNA expression
(Kroczek, J. Chromatogr. Biomed Appl., 618 (1-2) (1993) 133-145),
Western analysis of protein expression, or phenotypic analysis.
For some applications it will be useful to direct the instant GST
enzymes to different cellular compartments or to facilitate enzyme
secretion from a recombinant host cell. It is thus envisioned that
the chimeric genes described above may be further supplemented by
altering the coding sequences to encode enzymes with appropriate
intracellular targeting sequences such as transit sequences
(Keegstra, K., Cell 56:247-253 (1989)), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels,
J. J., Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53 (1991)), or
nuclear localization signals (Raikhel, N. Plant Phys. 100:1627-1632
(1992)) added and/or with targeting sequences that are already
present removed. While the references cited give examples of each
of these, the list is not exhaustive and more targeting signals of
utility may be discovered in the future that are useful in the
invention.
It may also be desirable to reduce or eliminate expression of the
genes encoding the instant GST enzymes in plants for some
applications. In order to accomplish this, chimeric genes designed
for co-suppression of the instant GST enzymes can be constructed by
linking the genes or gene fragments encoding the enzymes to plant
promoter sequences. Alternatively, chimeric genes designed to
express antisense RNA for all or part of the instant nucleic acid
fragments can be constructed by linking the genes or gene fragment
in reverse orientation to plant promoter sequences. Either the
co-suppression or antisense chimeric genes could be introduced into
plants via transformation wherein expression of the corresponding
endogenous genes are reduced or eliminated.
Plants transformed with the present GST genes will have a variety
of phenotypes corresponding to the various properties conveyed by
the GST class of proteins. Glutathione conjugation catalyzed by
GSTs is known to result in sequestration and detoxification of a
number of herbicides and other xenobiotics (Marrs et al., Annu.
Rev. Plant Physiol. Plant Mol. Biol. 47:127-58 (1996)) and thus
will be expected to produce transgenic plants with this phenotype.
Other GST proteins are known to be induced by various environmental
stresses such as salt stress (Roxas, et al., Stress tolerance in
transgenic seedlings that overexpress glutathione S-transferase,
Annual Meeting of the American Society of Plant Physiologists,
(August 1997), abstract 1574, Final Program, Plant Biology and
Supplement to Plant Physiology, 301), exposure to ozone (Sharma et
al., Plant Physiology, 105 (4) (1994) 1089-1096), and exposure to
industrial pollutants such as sulfur dioxide (Navari-Izzo et al.,
Plant Science 96 (1-2) (1994) 31-40). It is contemplated that
transgenic plants, tolerant to a wide variety of stresses, may be
produced by the present method by expressing foreign GST genes in
suitable plant hosts.
The instant GST enzymes produced in heterologous host cells,
particularly in the cells of microbial hosts, can be used to
prepare antibodies to the enzymes by methods well known to those
skilled in the art. The antibodies are useful for detecting the
enzymes in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant GST enzymes
are microbial hosts. Microbial expression systems and expression
vectors containing regulatory sequences that direct high level
expression of foreign proteins are well known to those skilled in
the art. Any of these could be used to construct chimeric genes for
production of the instant GST enzymes. These chimeric genes could
then be introduced into appropriate microorganisms via
transformation to provide high level expression of the enzymes.
Vectors or cassettes useful for the transformation of suitable host
cells are well known in the art. Typically the vector or cassette
contains sequences directing transcription and translation of the
relevant gene, a selectable marker, and sequences allowing
autonomous replication or chromosomal integration. Suitable vectors
comprise a region 5' of the gene which harbors transcriptional
initiation controls and a region 3' of the DNA fragment which
controls transcriptional termination. It is most preferred when
both control regions are derived from genes homologous to the
transformed host cell, although it is to be understood that such
control regions need not be derived from the genes native to the
specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive
expression of the genes encoding the GST enzymes in the desired
host cell are numerous and familiar to those skilled in the art.
Virtually any promoter capable of driving these genes is suitable
for the present invention including but not limited to CYC1, HIS3,
GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,
TPI (useful for expression in Saccharomyces); AOX1 (useful for
expression in Pichia); and lac, trp, .lambda.P.sub.L,
.lambda.P.sub.R, T7, tac, and trc (useful for expression in E.
coli).
Termination control regions may also be derived from various genes
native to the preferred hosts. Optionally, a termination site may
be unnecessary, however, it is most preferred if included.
An example of a vector for high level expression of the instant GST
enzymes in a bacterial host is provided (Example 5).
Additionally, the instant maize GST enzymes can be used as a
targets to facilitate design and/or identification of inhibitors of
the enzymes that may be useful as herbicides or herbicide
synergists. This is desirable because the enzymes described herein
catalyze the sulfhydryl conjugation of glutathione to compounds
toxic to the plant. Conjugation can result in detoxification of
these compounds. It is likely that inhibition of the detoxification
process will result in inhibition of plant growth or plant death.
Thus, the instant maize GST enzymes could be appropriate for new
herbicide or herbicide synergist discovery and design
All or a portion of the nucleic acid fragments of the instant
invention may also be used as probes for genetically and physically
mapping the genes that they are a part of, and as markers for
traits linked to expression of the instant enzymes. Such
information may be useful in plant breeding in order to develop
lines with desired phenotypes or in the identification of
mutants.
For example, the instant nucleic acid fragments may be used as
restriction fragment length polymorphism (RFLP) markers. Southern
blots (Maniatis) of restriction-digested plant genomic DNA may be
probed with the nucleic acid fragments of the instant invention.
The resulting banding patterns may then be subjected to genetic
analyses using computer programs such as MapMaker (Lander et at.,
(1987) Genomics 1:174-181) in order to construct a genetic map. In
addition, the nucleic acid fragments of the instant invention may
be used to probe Southern blots containing restriction
endonuclease-treated genomic DNAs of a set of individuals
representing parent and progeny of a defined genetic cross.
Segregation of the DNA polymorphisms is noted and used to calculate
the position of the instant nucleic acid sequence in the genetic
map previously obtained using this population (Botstein et al.,
(1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in
genetic mapping are described by Bernatzky, R. and Tanksley, S. D.
(Plant Mol. Biol. Reporter 4(1):37-41 (1986)). Numerous
publications describe genetic mapping of specific cDNA clones using
the methodology outlined above or variations thereof. For example,
F2 intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
Nucleic acid probes derived from the instant nucleic acid sequences
may also be used for physical mapping (i.e., placement of sequences
on physical maps; see Hoheisel et al., In: Nonmammalian Genomic
Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and
references cited therein).
In another embodiment, nucleic acid probes derived from the instant
nucleic acid sequences may be used in direct fluorescence in situ
hybridization (FISH) mapping. Although current methods of FISH
mapping favor use of large clones (several to several hundred KB),
improvements in sensitivity may allow performance of FISH mapping
using shorter probes.
A variety of nucleic acid amplification-based methods of genetic
and physical mapping may be carried out using the instant nucleic
acid sequences. Examples include allele-specific amplification,
polymorphism of PCR-amplified fragments (CAPS), allele-specific
ligation, nucleotide extension reactions, Radiation Hybrid Mapping
and Happy Mapping. For these methods, the sequence of a nucleic
acid fragment is used to design and produce primer pairs for use in
the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art.
In methods employing PCR-based genetic mapping, it may be necessary
to identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, this is generally not necessary for
mapping methods. Such information may be useful in plant breeding
in order to develop lines with desired starch phenotypes.
EXAMPLES
The present invention is further defined in the following Examples,
in which all parts and percentages are by weight and degrees are
Celsius, unless otherwise stated. It should be understood that
these Examples, while indicating preferred embodiments of the
invention, are given by way of illustration only. From the above
discussion and these Examples, one skilled in the art can ascertain
the essential characteristics of this invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various
usages and conditions.
GENERAL METHODS
Standard recombinant DNA and molecular cloning techniques used in
the Examples are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, pub. by Greene
Publishing Assoc. and Wiley-Interscience (1987).
Example 1
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
cDNA libraries representing mRNAs from various maize tissues were
prepared. The characteristics of the libraries are described in
Table 1.
TABLE 1 ______________________________________ cDNA Libraries From
Corn Tissues GST Library Class Clone Tissue
______________________________________ bms1 I bms1.pk0023.g8 Maize
BMS cell culture 1 day after subculture cs1 I cs1.pk0010.c5 Maize
leaf, sheath 5 wk plant Stratogene #837201 ceb1 I ceb1.pk0017.a5
Maize embryo cc71se III cc71se-a.pk0001.g2 Maize class II callus
tissue, somatic embryo formed, highly transformable cc71se III
cc71se-b.pk0014.b8 Maize class II callus tissue, somatic embryo
formed, highly transformable ceb5 III ceb5.pk0051.f8 Amplified
maize embryo 30 day cr1n III cr1n.pk0003.b1 Maize root from 7 day
seedlings grown in light normalized cr1n III cr1n.pk0014.g8 Maize
root from 7 day seedlings grown in light normalized m II
m.15.5.d06.sk20 Maize 15 day embryo library cr1n II cr1n.pk0040.e12
Maize root from 7 day seedlings grown in light normalized ceb5 III
ceb5.pk0049.a11 Amplified maize embryo 30 day cs1 III cs1.pk0059.e2
Maize leaf, sheath 5 wk plant Stratogene #837201 cbn2 gst I
cbn2.pk0032.d10 Corn (Zea mays L.) developing kernel two days after
pollination cr1n gst I cr1n.pk0164.g7 Corn (Zea mays L.) root from
7 day seedlings grown in light normalized cdt2 gst I
cdt2c.pk003.l15 Corn (Zea mays L.) developing tassel 2 csc1c gst I
csc1c.pk001.h7 Corn (Zea mays L., B73) 20 day seedling (germination
cold stress). The seedling appeared purple. p0110 gst I
p0110.cgsnt78r Corn (Zea mays L. B73) salacylic acid infiltrated
V3/V4 leaf tissue (minus midrib), screened 1 pool of A63 + SA 4 h;
A63 + SA 24 hr; and A63 + SA 7 days . p0121 gst I p0121.cfrmz42r
Corn (Zea mays L.) shank tissue collected from ears 5DAP, Screened
1 csi1n gst III csi1n.pk0034.a11 Corn (Zea mays L.) silk;
normalized from csi1 library cepe7 gst III cepe7.pk0028.g3 Corn
(Zea mays L.) epicotyl from 7 day old etiolated seedling cr1n gst
III cr1n.pk0167.d7 Corn (Zea mays L.) root from 7 day seedlings
grown in light normalized cco1 gst III cco1.pk0027.e4 Corn (Zea
mays L.) cob of 67 day old plants grown in green house cpj1c gst
III cpj1c.pk001.d21 Corn (Zea mays L.) pooled black mexican
sweetcorn treated with chemicals related to membrane ionic force
cse1c gst III cse1c.pk001.b8 Corn (Zea mays L.) seedling at V2
stage treated with Ethylene collected at 6 hr, 23 hr, 72 hr cr1s
gst III cr1s.pk010.f1 Corn (Zea mays L., Lh132) root from 7 day old
etiolated seedlings cpf1c gst III cpf1c.pk002.a13 Corn (Zea mays
L.) pooled black mexican sweetcorn treated with chemicals related
to protein synthesis cho1c gst III cho1c.pk004.c15 Corn (Zea mays
L., Alexho Synthetic High Oil) embryo 20 DAP cpi1c gst III
cpi1c.pk002.m4 Corn (Zea mays L.) pooled black mexican sweetcorn
treated with chemicals related to biochemical compound synthesis
chpc8 gst III chpc8.pk057.f10 Corn (Zea mays L., MBS847) 8 day old
shoot treated with PDO herbicide MK593 collected 8 hrs after
treatment. p0014 gst III p0014.ctu90r Corn (Zea mays L.) Leaf: Gene
uaz151 (G-Protein), 413-8, no genetic lesions are formed. C.
heterostrophus resistance, plant 3 ft tall, leaf 7 and leaf 8 p0006
gst III p0006.cbyvs55r Corn (Zea mays L.) Young shoot p0037 gst III
p0037.crwaf68r Corn (Zea mays L.) corn Root Worm infested V5 roots
p0032 gst III p0032.crcas61r Corn (Zea mays L.) Regenernerating
callus, 10 and 14 days after auxin removal. Hi-II callus 223a,
1129e 10 days. Hi-II callus 223a, 1129e 14 days p0088 gst III
p0088.c1rim45r Corn (Zea mays L.) Gene M1C07 (leucine-rich repeat),
family 3-B7. about one month after planting in green house p0126
gst III p0126.cn1ag50r Corn (Zea mays L.) Night harvested leaf
tissue; V8-V10 p0095 gst III p0095.cwsba73r Corn (Zea mays L.) Ear
leaf sheath, screened 1 Growth conditions: field; control or
untreated tissues Growth stage: 2-3 weeks after pollen shed; plants
were allowed to pollinate naturally p0125 gst III p0125.czaaj03r
Corn (Zea mays L.) Anther: Prophase I sceened 1
______________________________________
cDNA libraries were prepared in Uni-ZAP.TM. XR vectors according to
the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,
Calif.). The Uni-ZAP.TM. XR libraries were converted into plasmid
libraries according to the protocol provided by Stratagene. Upon
conversion, cDNA inserts were contained in the plasmid vector
pBluescript. cDNA inserts from randomly picked bacterial colonies
containing recombinant pBluescript plasmids were amplified via
polymerase chain reaction using primers specific for vector
sequences flanking the inserted cDNA sequences. Amplified insert
DNAs were sequenced in dye-primer sequencing reactions to generate
partial cDNA sequences (expressed sequence tags or "ESTs"; see
Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs
were analyzed using a Perkin Elmer Model 377 fluorescent
sequencer.
Example 2
Identification and Characterization of cDNA Clones
cDNAs encoding maize GST enzymes were identified by conducting
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al.,
(1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, SWISS-PROT protein sequence
database, EMBL, and DDBJ databases). The cDNA sequences obtained in
Example 1 were analyzed for similarity to all publicly available
DNA sequences contained in the "nr" database using the BLASTN
algorithm provided by the National Center for Biotechnology
Information (NCBI). The DNA sequences were translated in all
reading frames and compared for similarity to all publicly
available protein sequences contained in the "nr" database using
the BLASTX algorithm (Gish, W. and States, D. J. (1993) Nature
Genetics 3:266-272) provided by the NCBI. For convenience, the
P-value (probability) of observing a match of a cDNA sequence to a
sequence contained in the searched databases merely by chance as
calculated by BLAST are reported herein as "pLog" values, which
represent the negative of the logarithm of the reported P-value.
Accordingly, the greater the pLog value, the greater the likelihood
that the cDNA sequence and the BLAST "hit" represent homologous
proteins.
All comparisons were done using either the BLASTNnr or BLASTXnr
algorithms. The results of the BLAST comparisons are given in Table
2 and summarize the clones and the sequences to which they have the
most similarity. Table 2 displays data based on the BLASTNnr or
BLASTXnr algorithm with values reported in pLogs or Exprect values.
The Expect value estimates the statistical significance of the
match, specifying the number of matches, with a given score, that
are expected in a search of a database of this size absolutely by
chance. Each cDNA identified encodes at least a portion of either a
GST class I, II, or III. All isolated clones contain a fill length
open reading frame (ORF) with the exception of cc71se-a.pk0001.g2
which is only a partial clone. Example 5 describes the sequencing
strategy for the above described clones.
TABLE 2
__________________________________________________________________________
BLAST Results For Clones SEQ ID NO. GST Blast pLog Clone Class
Similarity Identified Base Peptide Algorithm Score*/E-Value* *
Citation
__________________________________________________________________________
bms1.pk0023.g8 I X79515.vertline.ZMGST27 Z. mays 1 2 Nnr 122.086
GST-27 mRNA for glutathione-S- transferase cs1.pk0010.c5 I
D17673.vertline.ATHERD13 3 4 Nnr 8.16 Arabidopsis thaliana mRNA for
glutathione S-transferase ceb1.pk0017.a5 I X78203.vertline.HMGST H.
muticus 5 6 Nnr 21.51 mRNA for glutathione S-transferase cc71se-
III (AF004358) glutathione S- 7 8 Nnr 16.48 a.pk0001.g2 transferase
TSI-1 (Aegilops squarrosa) cc71se- III D10861.vertline.RICORFC Rice
9 10 Nnr 14.96 b.pk0014.b8 mRNA for a protein related to chilling
tolerance. ceb5.pk0051.f8 III D1086T.vertline.RICORFC Rice 11 12
Nnr 40.44 mRNA for a protein related to chilling tolerance.
cr1n.pk0003.b1 III U80615.vertline.EGU80615 Eucalyptus 13 14 Nnr
24.70 globulus auxin-induced protein (EgPar) mRNA, complete cds
cr1n.pk0014.g8 III M16901.vertline.MZEGSTIB Maize 15 16 Nnr 5.85
glutathione S-transfer ase (GST-I) mRNA, complete cds
m.15.5.d06.sk20 II .vertline.M97702.vertline.DROGLUSTD 17 18 Nnr
3.63 Drosophila melanogaste r glutathione S-transferase gene.
cr1n.pk0040.e12 II 167970 (L05915) (GST1) gene 19 20 Xnr 42.03
product (Dianthus caryophyllus) ceb5.pk0049.a11 III
.vertline.Y12862.vertline.ZYMY12862 Zea Maize 21 22 Nnr 0.0 mRNA
for glutathione S-transferase cs1.pk0059.e2 III
D10861.vertline.RICORFC Rice mRNA for a 24 25 Nnr 41.03 protein
related to chilling tolerance. cbn2.pk0032.d10 gst I (AC005309)
glutathione s-transferase, 25 26 Xnr 4e-27 unpublished GST6
(Arabidopsis thaliana] cr1n.pk0164.g7 gst I (AC005309) glutathione
s-transferase, 27 28 Xnr 7e-37 unpublished GST6 [Arabidopsis
thaliana] cdec.pk003.115 gst I (AC005309) glutathione
s-transferase, 29 30 Xnr 4e-36 unpublished GST6 [Arabidopsis
thaliana] csc1c.pk001.h7 gst I (U70672) glutathione S-transferase
31 32 Xnr 8e-34 unpublished [Arabidopsis thaliana] p0110.cgsnt78r
gst I P46420.vertline.GTH4.sub.-- MAIZE 33 34 Xnr 1e-97 Plant Mol.
Biol. 26 (6), GLUTSTHION S-TRANSFERASE 1855-1866 (1994) IV (GST-IV)
(GS-27) p0121.cfrmz42r. gst I P42761.vertline.GTH3.sub.-- ARATH 35
36 Xnr 3e-28 FEBS Lett. 335 (2), 189-192 GLUTETHIONE S-TRANSFERASE
(1993) ERD13 (CLASS PHI) csi1n.pk0034.a11 gst III
Q03664.vertline.GTX3.sub.-- TOBAC PROBABLE 37 38 Xnr 2e-51 Plant
Mol. Biol. 16 (6), 983-998 GLUTATHIONE S-TRANSFER ASE (1991)
(AUXIN-INDUCED PROTEIN PCNT103) cepe7.pk0028.g3 gst III (AF004358)
glutathione S-transferase 39 40 Xnr 9e-50 Plant Physiol. 114,
1461-1470 TSI-1 [Aegilops squarrosa] (1997) cr1n.pk0167.d7 gst III
(AF004358) glutathione S-transferase 41 42 Xnr 8e-58 Plant
Physiol.
114, 1461-1470 TSI-1 [Aegilops squarrosa] (1997) cco1.pk0027.e4 gst
III (Y12862) glutathione transferase [Zea 43 44 Xnr 2e-77 JOURNAL
Plant Mol. Biol. 36, mays] 75-87 (1998) cpj1c.pk001.d21 gst III
.vertline.Q03662.vertline.GTX1.sub.-- TOBAC 45 46 Xnr 1e-53 Plant
Mol. Biol. 16 (6), 983-998 PROBABLE GLUTATHIONE (1991)
S-TRANSFERASE (AUXIN- INDUCED PROTEIN PGNT1/PCNT110) cse1c.pk001.b8
gst III (AF004358) glutathione S-transferase 47 48 Xnr 3e-62 Plant
Physiol. 114, 1461-1470 TSI-1 [Aegilops squarrosa] (1997)
cr1s.pk010.f1 gst III P32110.vertline.GTX6.sub.-- SOYBN PROBABLE 49
50 Xnr 4e-49 Mol. Cell. Biol. 8 (3), GLUTATHIONE S-TRANSFERASE
1113-1122 (1988) (HEAT SHOCK PROTEIN 26A) cpf1c.pk002.a13 gst III
Q03662.vertline.GTX1.sub.-- TOBAC PROBABLE 51 52 Xnr 6e-47 Plant
Mol. Biol. 16 (6), GLUTATHIONE S-TRANSFERASE 983-998 (1991)
(AUXIN-INDUCED PROTEIN PGNT1/PCNT110) cho1c.pk004.c15 gst III
(AF004358) glutathione S-transferase 53 54 Xnr 1e-59 Plant Physiol.
114, 1461-1470 TSI-1 [Aegilops squarrosa] (1997) cpi1c.pk002.m4 gst
III (AF051214) probable glutathione 55 56 Xnr 7e-45 Genetics 149
(2), 1089-1098 S-transferase [Picea mariana] (1998) chpc8.pk057.f10
gst III (AJ010449) glutathione transferase 57 58 Xnr 1e-62
unpublished [Alopecurus myosuroide s] p0014.ctu90r gst III
(AJ010448) glutathione transferase 59 60 Xnr 8e-55 unpublished
[Alopecurus myosuroides] p0006.cbyvs55r gst III (AF051214) probable
glutathione 61 62 Xnr 4e-51 Genetics 149 (2), 1089-1098
S-transferase [Picea mariana] (1998) p0037.crwaf68r gst III
(AF004358) glutathione S-transferase 63 64 Xnr 2e-77 Plant Physiol.
114, 1461-1470 TSI-1 [Aegilops squrrosa] (1997) p0032.crcas61r gst
III P32110 GTX6.sub.-- SOYBN PROBABLE 65 66 Xnr 3e-48 Mol. Cell.
Biol. 8 (3), GLUTATHIONE S-TRANSFERASE 1113-1122 (1988) (HEAT SHOCK
PROTEIN 26A) p0088.c1rim45r gst III (AF004358) glutathione
S-transferase 67 68 Xnr 3e-53 Plant Physiol. 114, 1461-1470 TSI-1
[Aegilops squarrosa] (1997) p0126.cn1ag50r gst III (AF004358)
glutathione S-transferase 69 70 Xnr 4e-52 Plant Physiol. 114,
1461-1470 TSI-1 [Aegilops squarrosa] (1997) p0095.cwsba73r gst III
(AF004358) glutathione S-transferase 71 72 Xnr 2e-57 Plant Physiol.
114, 1461-1470 TSI-1 [Aegilops squarrosa] (1997) p0125.czaaj03r gst
III (AF004358) glutathione S-transferase 73 74 Xnr 7E-63 Plant
Physiol. 114, 1461-1470 TSI-1 [Aegilops squarrosa] (1997)
__________________________________________________________________________
*Plog represents the negative of the logarithm of the reported
Pvalue **Expect value estimates the statistical significance of the
match, specifying the number of matches, with a given score, that
are expected i a search of a database of this size absolutely by
chance.
Example 3
Expression of Chimeric Genes Encoding Maize GST Enzymes in Maize
Cells (Monocotyledon)
A chimeric gene comprising a cDNA encoding a maize GST enzyme in
sense orientation can be constructed by polymerase chain reaction
(PCR) of the cDNA clone using appropriate oligonucleotide primers.
Cloning sites (NcoI or SmaI) can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the digested vector pML103 as described below.
Amplification is then performed in a 100 .mu.L volume in a standard
PCR mix consisting of 0.4 mM of each oligonucleotide and 0.3 pM of
target DNA in 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2,
200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTP and 0.025 unit
DNA polymerase. Reactions are carried out in a Perkin-Elmer Cetus
Thermocycler.TM. for 30 cycles comprising 1 min at 95.degree. C., 2
min at 55.degree. C. and 3 min at 72.degree. C., with a final 7 min
extension at 72.degree. C. after the last cycle. The amplified DNA
is then digested with restriction enzymes NcoI and SmaI and
fractionated on a 0.7% low melting point agarose gel in 40 mM
Tris-acetate, pH 8.5, 1 mM EDTA. The appropriate band can be
excised from the gel, melted at 68.degree. C. and combined with a
4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has
been deposited under the terms of the Budapest Treaty at ATCC
(American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md. 20852), and bears accession number ATCC 97366. The DNA segment
from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the
maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3'
end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega
Corp 7113 Benhart Dr, Raleigh, N.C.). Vector and insert DNA can be
ligated at 15.degree. C. overnight, essentially as described
(Maniatis). The ligated DNA may then be used to transform E. coli
XL1-Blue (Epicurian Coli XL-1; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method DNA Sequencing Kit; U.S. Biochemical). The
resulting plasmid construct would comprise a chimeric gene
encoding, in the 5' to 3' direction, the maize 27 kD zein promoter,
a cDNA fragment encoding a plant GST enzyme, and the 10 kD zein 3'
region. The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132 (Indiana Agric. Exp. Station,
Ind., USA). The embryos are isolated 10 to 11 days after
pollination when they are 1.0 to 1.5 mm long. The embryos are then
placed with the axis-side facing down and in contact with
agarose-solidified N6 medium (Chu et al., (1975) Sci. Sin. Peking
18:659-668). The embryos are kept in the dark at 27.degree. C.
Friable embryogenic callus consisting of undifferentiated masses of
cells with somatic proembryoids and embryoids borne on suspensor
structures proliferates from the scutellum of these immature
embryos. The embryogenic callus isolated from the primary explant
can be cultured on N6 medium and sub-cultured on this medium every
2 to 3 weeks. The plasmid, p35S/Ac (obtained from Dr. Peter Eckes,
Hoechst Ag, v Frankfurt, Germany) may be used in transformation
experiments in order to provide for a selectable marker. This
plasmid contains the Pat gene (see European Patent Publication 0
242 236) which encodes phosphinothricin acetyl transferase (PAT).
The enzyme PAT confers resistance to herbicidal glutamine
synthetase inhibitors such as phosphinothricin. The pat gene in
p35S/Ac is under the control of the 35S promoter from Cauliflower
Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3M
region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The particle bombardment
method (Klein et al., (1987) Nature 327:70-73) may be used to
transfer genes to the callus culture cells. According to this
method, gold particles ((1 .mu.m in diameter) are coated with DNA
using the following technique. Ten ug of plasmid DNAs are added to
50 uL of a suspension of gold particles (60 mg per mL). Calcium
chloride (50 .mu.L of a 2.5 M solution) and spermidine free base
(20 .mu.L of a 1.0 M solution) are added to the particles. The
suspension is vortexed during the addition of these solutions.
After 10 minutes, the tubes are briefly centrifuged (5 sec at
15,000 rpm) and the supernatant removed. The particles are
resuspended in 200 .mu.L of absolute ethanol, centrifuged again and
the supernatant removed. The ethanol rinse is performed again and
the particles resuspended in a final volume of 30 uL of ethanol. An
aliquot (5 .mu.L) of the DNA-coated gold particles can be placed in
the center of a flying disc (Bio-Rad Labs, 861 Ridgeview Dr,
Medina, Ohio). The particles are then accelerated into the corn
tissue with a PDS-1000/He (Bio-Rad Labs, 861 Ridgeview Dr, Medina,
Ohio), using a helium pressure of 1000 psi, a gap distance of 0.5
cm and a flying distance of 1.0 cm. For bombardment, the
embryogenic tissue is placed on filter paper over
agarose-solidified N6 medium. The tissue is arranged as a thin lawn
and covered a circular area of about 5 cm in diameter. The petri
dish containing the tissue can be placed in the chamber of the
PDS-1000/He approximately 8 cm from the stopping screen. The air in
the chamber is then evacuated to a vacuum of 28 inches of Hg. The
macrocarrier is accelerated with a helium shock wave using a
rupture membrane that bursts when the He pressure in the shock tube
reaches 1000 psi. Seven days after bombardment the tissue can be
transferred to N6 medium that contains gluphosinate (2 mg per
liter) and lacks casein or proline. The tissue continues to grow
slowly on this medium. After an additional 2 weeks the tissue can
be transferred to fresh N6 medium containing gluphosinate. After 6
weeks, areas of about 1 cm in diameter of actively growing callus
can be identified on some of the plates containing the
glufosinate-supplemented medium. These calli may continue to grow
when sub-cultured on the selective medium. Plants can be
regenerated from tie transgenic callus by first transferring
clusters of tissue to N6 medium supplemented with 0.2 mg per liter
of 2,4-D. After two weeks the tissue can be transferred to
regeneration medium (Fromm et al., (1990) Bio/Technology
8:833-839).
Example 4
Expression of Chimeric Genes in Tobacco Cells (Dicotyledon)
Cloning sites (XbaI or SmaI) can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the digested vector pBI121 (Clonetech Inc., 6500
Donlon Rd, Somis, Calif.) or other appropriate transformation
vector. Amplification could be performed as described above and the
amplified DNA would then be digested with restriction enzymes XbaI
and SmaI and fractionated on a 0.7% low melting point agarose gel
in 40 mM-Tris-acetate, pH 8.5, 1 mM EDTA. The appropriate band can
be excised from the gel, melted at 68.degree. C. and combined with
a 13 kb XbaI-SmaI fragment of the plasmid pBI121 and handled as in
Example 3. The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, right border
region, the nos promoter linked to the NPT II gene and a nos
terminator region followed by a cauliflower mosaic virus 35S
promoter linked to a cDNA fragment encoding a plant GST enzyme and
the nos terminator 3' region flanked by the left border region. The
resulting plasmid could be mobilized into the Agrobacterium strain
LBA4404/pAL4404 (Hoekema et al. Nature 303:179-180, (1983) using
triparental matings (Ruvkin and Ausubel, Nature 289:85-88, (1981)).
The resulting Agrobacterium strains could be then cocultivated with
protoplasts (van den Elzen et al. Plant Mol. Biol, 5:149-154
(1985)) or leaf disks (Horsch et al. Science 227:1229-1231, (1985))
of Nicotiana tabacum cv Wisconsin 38 and kanamycin-resistant
transformants would be selected. Kanamycin-resistant transformed
tobacco plants would be regenerated.
Example 5
Expression of Chimeric Genes in Microbial Cells and Purification of
Gene Product
Example 5 illustrates the expression of isolated full length genes
encoding either class I, II or III GST proteins in E. coli.
All clones listed in Table 2 were selected on the basis of homology
to known GSTs using the BLAST algorithm as described in Example 2.
Plasmid DNA was purified using QIAFilter cartridges (Qiagen. Inc.,
9600 De Soto Ave, Chatsworth, Calif.) according to the
manufacturer's instructions. Sequence was generated on an ABI
Automatic sequencer using dye terminator technology (U.S. Pat. No.
5,366,860; EP 272007) using a combination of vector and
insert-specific primers. Sequence editing was performed in either
DNAStar (DNA, Star Inc.) or the Wisconsin GCG program (Wisconsin
Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).
All sequences represent coverage at least two times in both
directions.
cDNA from the clones bms1.pk0023.g8, cs1.pk0010.c5, ceb1.pk0017.a5,
m.15.5.d06.sk20, ceb5.pk0049.a11, ceb5.pk0051.f8, and
cs1.pk0059.e2, encoding the instant maize GST enzymes were inserted
into the ligation independent cloning (LIC) pET30 vector (Novagen,
Inc., 597 Science Dr, Madison, Wis.) under the control of the T7
promoter, according to the manufacturer's instructions (see Novagen
publications "LIC Vector Kits", publication number TB163 and U.S.
Pat. No. 4,952,496). The vector was then used to transform
BL21(DE3) competent E. coli hosts. Primers with a specific 3'
extension designed for ligation independent cloning were designed
to amplify the GST gene Maniatis). Amplification products were
gel-purified and annealed into the LIC vector after treatment with
T4 DNA polymerase (Novagen). Insert-containing vectors were then
used to transform NovaBlue competent E. coli cells and
transformants were screened for the presence of viable inserts.
Clones in the correct orientation with respect to the T7 promoter
were transformed into BL21(DE3) competent cells (Novagen) and
selected on LB agar plates containing 50 .mu.g/mL kanamycin.
Colonies arising from this transformation were grown overnight at
37.degree. C. in Lauria Broth to OD 600=0.6 and induced with 1 mM
IPTG and allowed to grow for an additional two hours. The culture
was harvested, resuspended in binding buffer, lysed with a French
press and cleared by centrifugation.
Expressed protein was purified using the HIS binding kit (Novagen)
according to the manufacturer's instructions. Purified protein was
examined on 15-20% SDS Phast Gels (Bio-Rad Laboratories, 861
Ridgeview Dr, Medina, Ohio) and quantitated spectrophotometrically
using BSA as a standard. Protein data is tabulated below in Table
3.
TABLE 3 ______________________________________ Protein Expression
Data CLONE OD.280 ______________________________________
bms1.pk0023.g8 0.57 cs1.pk0010.c5 0.53 ceb1.pk0017.a5 0.50
m.15.5.d06.sk20 0.39 ceb5.pk0049.a11 2.06 ceb5.pk0051.f8 1.30
cs1.pk0059.e2 1.45 ______________________________________
Example 6
Screening of Expressed GST Enzymes for Substrate Metabolism
The GST enzymes, expressed and purified as described in Example 5
were screened for their ability to metabolize a variety of
substrates. Substrates tested included the three herbicide
electrophilic substrates chlorimuron ethyl, alachlor, and Atrazine,
and four model electrophilic substrates,
1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, t-stilbene
oxide, and 1,2-epoxy-3-(p-nitrophenoxy)propane. The enzymes were
purified as described in Example 5 and used in the following
assay.
For each enzyme, the conjugation reaction with each electrophilic
substrate was performed by incubating 0.3 to 30 .mu.g enzyme in 0.1
M MOPS (pH 7.0) containing 0.4 mM of the electrophilic substrate.
The reaction was inititated by the addition of glutathione to a
final concentration of 4 mM. After 5 to 30 min, the reaction was
terminated by the addition of 45 .mu.L acetonitrile, microfuged for
10 min to remove precipitated protein, and then the supernatent was
removed and added to 65 .mu.l of water. This sample was
chromatographed on a Zorbax C8 reverse phase HPLC column (3 .mu.m
particle size, 6.2 mm.times.8 cm) using a combination of linear
gradients (flow=1.5 mL/min) of 1% H.sub.3 PO.sub.4 in water
(solvent A) and 1% H.sub.3 PO.sub.4 in acetonitrile. The gradient
started with 5% solvent B, progressing from 5% to 75% solvent B
between 1 and 10 min, and from 75% to 95% solvent B between 10 and
12 min. Control reactions without enzyme were performed to correct
for uncatalyzed reaction. Quantitation of metabolites were based on
an assumption that the extinction coefficient of the conjugate was
identical to that of the electrophilic substrate.
Table 4 shows the activity of each enzyme measured in
nmol.min.sup.-1.mg.sup.-1 with the seven different substrates.
Activities are related to the activities of the known and
previously isolated and purified GST enzymes, BZ-II (Marrs et al.,
Nature 375:397-400 (1995)), pIN2-1 (Hershey et al., Plant Molecular
Biology 17:679-690, (1991)), GST-I, GST-III, and GST-IV,
collectively described in Shah et al., Plant Mol Biol 6,
203-211(1986); Jepson et al., Plant Mol Biol 26:1855-1866, (1994);
Moore et al., Nucleic Acids Res 14:7227-7235 (1986); and Holt et
al., Planta 196:295-302, (1995).
TABLE 4
__________________________________________________________________________
Chlor- 1,2-epoxy-3- GST Imuron- Ethacrynic t-Stilbene
(p-nitrophenoxy) GST Name Class Ethyl Alachlor Atrazine CDNB Acid
Oxide propane
__________________________________________________________________________
cs1.pk0059.e2 III 0.1 8 0.02 1348 20 1.25 43 ceb5.pk0049.a11 III
0.4 18 0.01 3939 102 0.01 30 ceb5.pk0051.f8 III 1.9 27 0.08 2136
117 0.02 14 BZ-II llI 0.2 0 0.00 15 23 0.05 0 ceb1.pk0017.a5 I 0.1
0 0.00 15 5 0.00 0 cs1.pk0010.c5 I 0.1 0 0.00 30 9 0.00 0
bms1.pk0023.g8 I 0.2 0 0.00 15 13 0.00 0 GST-IV i 0.3 1 0.00 15 13
0.00 0 GST-I I 0.4 77 0.60 46485 32 0.98 92 GST-III I 0.3 3 0.05
1803 1 0.31 28 m.15.5.d06.sk20 II 0.1 0 0.00 45 17 0.00 1 pIN2-1 IV
0 0 -- 15 -- -- --
__________________________________________________________________________
__________________________________________________________________________
# SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 74 - -
<210> SEQ ID NO 1 <211> LENGTH: 844 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 1 - -
cggcacgagc aatggcgccg ccgatgaagg tgtacgggtg ggccgtgtcg cc -
#gtggatgg 60 - - cgcgcgcgct ggtgtgtctg gaggaggccg gcgccgacta
cgagatcgtc cc - #catgagca 120 - - ggtgtggcgg cgaccaccgc cggccggagc
acctcgccaa aaacccgttc gg - #tgaaatcc 180 - - cagttttaga ggacggtgat
ctcacgctct accaatcacg cgccatcgca cg - #gtacgtcc 240 - - tccgcaagct
caagccagag ctcctccgcg aaggcgacct cgaggggtcg gc - #gatggtgg 300 - -
acgcgtggat ggaggtggaa gcccaccaca tggagccggc cctgtggccc at -
#catccgcc 360 - - acagcatcat cggccagtac gtcggccgcg agcgcgacca
ccaggccgtc at - #cgacgaga 420 - - acctcgacag gctgaggaag gtgctgccgg
cgtacgaggc gaggctgtcc gt - #ctgcaagt 480 - - acctggtggg ggacgacatc
agcgccgccg acctctgcca cttcggcttc at - #gcgctact 540 - - tcatggccac
ggagtacgcc ggcttggtgg acgcgtaccc gcacgtcaag gc - #ctggtggg 600 - -
acgcgctgct ggcgaggccc tcggtgcaga aggtcatggc aggcatgccg cc -
#ggattttg 660 - - ggtacgccag cgggaacata ccataggcta gaagcggtgg
gcgtccgtca tt - #ctgcagat 720 - - ctgaggtctc tgaacctcag cgtttccgat
aaacatgcat gctttatgta ct - #gtttaaaa 780 - - aacaaacctg attggtgcag
ggtattttag tcctcttaaa aaaaaaaaaa aa - #aaaaaaaa 840 - - aaaa - # -
# - # 844 - - - - <210> SEQ ID NO 2 <211> LENGTH: 224
<212> TYPE: PRT <213> ORGANISM: maize - - <400>
SEQUENCE: 2 - - Met Ala Pro Pro Met Lys Val Tyr Gly Trp Al - #a Val
Ser Pro Trp Met 1 5 - # 10 - # 15 - - Ala Arg Ala Leu Val Cys Leu
Glu Glu Ala Gl - #y Ala Asp Tyr Glu Ile 20 - # 25 - # 30 - - Val
Pro Met Ser Arg Cys Gly Gly Asp His Ar - #g Arg Pro Glu His Leu 35
- # 40 - # 45 - - Ala Lys Asn Pro Phe Gly Glu Ile Pro Val Le - #u
Glu Asp Gly Asp Leu 50 - # 55 - # 60 - - Thr Leu Tyr Gln Ser Arg
Ala Ile Ala Arg Ty - #r Val Leu Arg Lys Leu 65 - # 70 - # 75 - # 80
- - Lys Pro Glu Leu Leu Arg Glu Gly Asp Leu Gl - #u Gly Ser Ala Met
Val 85 - # 90 - # 95 - - Asp Ala Trp Met Glu Val Glu Ala His His Me
- #t Glu Pro Ala Leu Trp 100 - # 105 - # 110 - - Pro Ile Ile Arg
His Ser Ile Ile Gly Gln Ty - #r Val Gly Arg Glu Arg 115 - # 120 - #
125 - - Asp His Gln Ala Val Ile Asp Glu Asn Leu As - #p Arg Leu Arg
Lys Val 130 - # 135 - # 140 - - Leu Pro Ala Tyr Glu Ala Arg Leu Ser
Val Cy - #s Lys Tyr Leu Val Gly 145 1 - #50 1 - #55 1 - #60 - - Asp
Asp Ile Ser Ala Ala Asp Leu Cys His Ph - #e Gly Phe Met Arg Tyr 165
- # 170 - # 175 - - Phe Met Ala Thr Glu Tyr Ala Gly Leu Val As - #p
Ala Tyr Pro His Val 180 - # 185 - # 190 - - Lys Ala Trp Trp Asp Ala
Leu Leu Ala Arg Pr - #o Ser Val Gln Lys Val 195 - # 200 - # 205 - -
Met Ala Gly Met Pro Pro Asp Phe Gly Tyr Al - #a Ser Gly Asn Ile Pro
210 - # 215 - # 220 - - - - <210> SEQ ID NO 3 <211>
LENGTH: 999 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 3 - - atcaccattc ttcatccctc gttgtcatct
cacagcttgg gctagagacc aa - #acaaacca 60 - - aagggaagca tggcagcagg
cctgcaagtg tttggccagc cggcgtctac tg - #atgttgcc 120 - - agggttctga
cgtgcctgtt tgagaagaaa ttggagtttg agcttgtccg ca - #ttgataca 180 - -
tttaagacac atcacaggct tcctgagttc atcaggctgc gggatccgaa tg -
#ggcaagtg 240 - - accttcaagc atggcgacaa aacccttgtt gattcaaggg
acatatgccg gt - #acgtttgc 300 - - aaccagtttc caaattacgg aaacaagagc
ctctatggat ctggtgctct ag - #aacgggca 360 - - tcgatagaac agtggctcca
ggcagaagcc cagaactttg gccctcccag ct - #ctgcgctt 420 - - gtgtttcagc
tggcgttcgt tccgcacctc agtcacctgg gcgttcgtca gg - #accctgct 480 - -
gttattgctg aaaacgagga caaactgaag caggttcttg atgtttacga cg -
#aaatactc 540 - - tccaagaacg agtacctggc tggtgatgag ttcaccctgg
ccgacctgtc tc - #accttccg 600 - - aactcgcact acatcgtaaa caccgagaga
ggaaggaagc tcttcaccaa ca - #agaagaat 660 - - gtggcgaaat ggtatgacag
gctctcgaag cgcgagacat gggtgcaggt cg - #tcaagatg 720 - - cagaaggaac
atcctggtgc gttcaagtaa tggcttgtct tggggagttg tg - #agtatggc 780 - -
ttcatcgtcc gtgttggtct ggctcatcag tgttaaaagc ccatcagtgt cg -
#tcaaccag 840 - - aataatgtga agcccaactg tgatgtatgg tctttttttt
ttaaaagcgc at - #ttgtaaac 900 - - tattggctat ttcttgcacg tgccaattca
tcgtcacata taaaataaac tg - #tatctttg 960 - - accttgtgtc atgtacgcaa
aaaaaaaaaa aaaaaaaaa - # - # 999 - - - - <210> SEQ ID NO 4
<211> LENGTH: 226 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 4 - - Met Ala Ala Gly Leu Gln Val
Phe Gly Gln Pr - #o Ala Ser Thr Asp Val 1 5 - # 10 - # 15 - - Ala
Arg Val Leu Thr Cys Leu Phe Glu Lys Ly - #s Leu Glu Phe Glu Leu 20
- # 25 - # 30 - - Val Arg Ile Asp Thr Phe Lys Thr His His Ar - #g
Leu Pro Glu Phe Ile 35 - # 40 - # 45 - - Arg Leu Arg Asp Pro Asn
Gly Gln Val Thr Ph - #e Lys His Gly Asp Lys 50 - # 55 - # 60 - -
Thr Leu Val Asp Ser Arg Asp Ile Cys Arg Ty - #r Val Cys Asn Gln Phe
65 - # 70 - # 75 - # 80 - - Pro Asn Tyr Gly Asn Lys Ser Leu Tyr Gly
Se - #r Gly Ala Leu Glu Arg 85 - # 90 - # 95 - - Ala Ser Ile Glu
Gln Trp Leu Gln Ala Glu Al - #a Gln Asn Phe Gly Pro 100 - # 105 - #
110 - - Pro Ser Ser Ala Leu Val Phe Gln Leu Ala Ph - #e Val Pro His
Leu Ser 115 - # 120 - # 125 - - His Leu Gly Val Arg Gln Asp Pro Ala
Val Il - #e Ala Glu Asn Glu Asp 130 - # 135 - # 140 - - Lys Leu Lys
Gln Val Leu Asp Val Tyr Asp Gl - #u Ile Leu Ser Lys Asn 145 1 - #50
1 - #55 1 - #60 - - Glu Tyr Leu Ala Gly Asp Glu Phe Thr Leu Al - #a
Asp Leu Ser His Leu 165 - # 170 - # 175 - - Pro Asn Ser His Tyr Ile
Val Asn Thr Glu Ar - #g Gly Arg Lys Leu Phe 180 - # 185 - # 190 - -
Thr Asn Lys Lys Asn Val Ala Lys Trp Tyr As - #p Arg Leu Ser Lys Arg
195 - # 200 - # 205 - - Glu Thr Trp Val Gln Val Val Lys Met Gln Ly
- #s Glu His Pro Gly Ala 210 - # 215 - # 220 - - Phe Lys 225 - - -
- <210> SEQ ID NO 5 <211> LENGTH: 901 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 5 - -
ccccagcggc ggcgaggcga tggcggcgcc tgtgacggtg tacggaccga tg -
#ctctcacc 60 - - agctgtggcc cgcgtggcgg cctgcctcct ggagaaggac
gtgccgttcc ag - #atcgagcc 120 - - ggtggacatg tccaagggcg agcacaagtc
gccgtccttc ctcaagctcc ag - #cccttcgg 180 - - acaggtccct gccttcaagg
accacctcac aaccgtcttt gagtcaaggg ct - #atttgccg 240 - - ttacatatgc
gaccagtatg cggactctgg taatcaggcc ctcttcggca ag - #aaagaaga 300 - -
cggcgcggtt ggccgcgctg ccattgaaca gtggatagag tctgaaggcc ag -
#agctttaa 360 - - cccaccgagc ttggctatta tcttccagct cgcatttgca
ccgatgatgg gc - #cggaccac 420 - - tgacctggct gtggttgagc araatgaagc
gaagcttgcg aaggtgcttg at - #gtgtatga 480 - - ccaacggctg ggggagagcc
agtattttgc tggtgatgat ttctcccctg gc - #cgaccttg 540 - - tgcacttgcc
caatgcagat ttccttgtga acagaaccag caaggctggc tt - #gatcaccg 600 - -
agagaaagaa tcttgctaga tggtgggatg atgtctcgtc ccgacctgca tg -
#gaaaaagg 660 - - tcactgagat gcagagcacg ccgaggccct cttagagctt
ttttttgggt tt - #ctttgagc 720 - - agcttctgat ggcaattagt tgcattctcc
ttgttttgtc atcaagtcct tg - #tctgtacc 780 - - gtttcctgtt ctcttattta
tcggtcttaa ttcttgatct atgtatggtt tg - #gatctgtt 840 - - cttctggtcc
tttagtttat ataagtacct acaattcttc aaaaaaaaaa aa - #aaaaaaaa 900 - -
a - # - # - # 901 - - - - <210> SEQ ID NO 6 <211>
LENGTH: 199 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 6 - - Met Ala Ala Pro Val Thr Val Tyr Gly Pro
Me - #t Leu Ser Pro Ala Val 1 5 - # 10 - # 15 - - Ala Arg Val Ala
Ala Cys Leu Leu Glu Lys As - #p Val Pro Phe Gln Ile 20 - # 25 - #
30 - - Glu Pro Val Asp Met Ser Lys Gly Glu His Ly - #s Ser Pro Ser
Phe Leu
35 - # 40 - # 45 - - Lys Leu Gln Pro Phe Gly Gln Val Pro Ala Ph -
#e Lys Asp His Leu Thr 50 - # 55 - # 60 - - Thr Val Phe Glu Ser Arg
Ala Ile Cys Arg Ty - #r Ile Cys Asp Gln Tyr 65 - # 70 - # 75 - # 80
- - Ala Asp Ser Gly Asn Gln Ala Leu Phe Gly Ly - #s Lys Glu Asp Gly
Ala 85 - # 90 - # 95 - - Val Gly Arg Ala Ala Ile Glu Gln Trp Ile Gl
- #u Ser Glu Gly Gln Ser 100 - # 105 - # 110 - - Phe Asn Pro Pro
Ser Leu Ala Ile Ile Phe Gl - #n Leu Ala Phe Ala Pro 115 - # 120 - #
125 - - Met Met Gly Arg Thr Thr Asp Leu Ala Val Va - #l Glu Gln Asn
Glu Ala 130 - # 135 - # 140 - - Lys Leu Ala Lys Val Leu Asp Val Tyr
Asp Gl - #n Arg Leu Gly Glu Ser 145 1 - #50 1 - #55 1 - #60 - - Gln
Tyr Phe Ala Gly Asp Asp Phe Ser Pro Gl - #y Arg Pro Cys Ala Leu 165
- # 170 - # 175 - - Ala Gln Cys Arg Phe Pro Cys Glu Gln Asn Gl - #n
Gln Gly Trp Leu Asp 180 - # 185 - # 190 - - His Arg Glu Lys Glu Ser
Cys 195 - - - - <210> SEQ ID NO 7 <211> LENGTH: 458
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 7 - - gcgcgtcgga ggagctccac ggcgtcaggc ccttcgaccc
cgagcggact cc - #gctgctgg 60 - - cggcgtggtc ggagcgcttc ggcgcgctgg
atgccgtcca gacggtgatg cc - #cgacgtcg 120 - - gcaggctgct cgagttcggc
aaggcgttga tggcacgtct ggcggctgcg gc - #cgccgccg 180 - - gtgcaagcaa
taactgaaga gggcatggtg tatccgtcat gtgtttcagg tt - #ttcgtata 240 - -
gtgaacaaaa aaggaaaaaa taatgctagc tacgcatcgg aacgcggctt tg -
#tgctttgc 300 - - cgtctcgccg ttagttcagc ttatgtgatg tgagtgttgc
cgtgcatgtg tg - #tgttactt 360 - - cagatgtatc ctgttcggtt cagtgattat
atggaacatt ttattttggt tg - #gataaaaa 420 - - aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaa - # - # 458 - - - - <210> SEQ ID NO 8
<211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 8 - - Ala Ser Glu Glu Leu His Gly
Val Arg Pro Ph - #e Asp Pro Glu Arg Thr 1 5 - # 10 - # 15 - - Pro
Leu Leu Ala Ala Trp Ser Glu Arg Phe Gl - #y Ala Leu Asp Ala Val 20
- # 25 - # 30 - - Gln Thr Val Met Pro Asp Val Gly Arg Leu Le - #u
Glu Phe Gly Lys Ala 35 - # 40 - # 45 - - Leu Met Ala Arg Leu Ala
Ala Ala Ala Ala Al - #a Gly Ala Ser Asn Asn 50 - # 55 - # 60 - - -
- <210> SEQ ID NO 9 <211> LENGTH: 911 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 9 - -
gcaaggtcga catgtcgtct ccgccgccgg tgaagctgat cggcttcttc gg -
#cagcccgt 60 - - acgcgttccg cgcggaggcg gcgctgtgcc tgaaaggcgt
gccgtacgag ct - #gatcctgg 120 - - aggacctgtt cggcagcaag agcgagctcc
tgctccacca caaccccgtg ca - #caagaagg 180 - - tgcccgtgct cctccacggc
gacggccggg ccatctccga gtccctcgtc at - #cgccgagt 240 - - acgtcgacga
ggccttcgac gggccgccgc tgctccccgc cgacccctac gc - #gcgcgccg 300 - -
ccgcccgctt ctgggccgac ttcatcgaga ccaggctcac caagcccttc tt -
#catggcga 360 - - tctgggtgga ggagcgcgac gcgcggctgc ggttcgagga
ggaggccaag ga - #gctcgtgg 420 - - cgctgctgga ggcgcagctc gagggaaaga
ggttcttcgc cggcgacagg cc - #ggggtacc 480 - - tcgacgtggc cgcgtccgcg
ctcgggccct ggcgcagcgt catcgaggag ct - #caacggtg 540 - - tggcgctgct
cagcgaggat gaccacccca acctgtgccg gtggaccagg ga - #ctactgcg 600 - -
ccttcgaggc tctcaagccg tgcatgccgg atcgggagaa gctcctcgcc ta -
#cttcacta 660 - - agaacttcga caggtacaag gcggccgtca atgcgacgct
atcgcagtcg ca - #gcagtaat 720 - - aactgcccaa ctgggtacgc ctctgcccgg
ccgtatggcg ggcgtttctt tt - #tttctttc 780 - - ttcagaataa cgtagctgtg
cccagtactc atgttttcaa ttctgcaaag tg - #caaaccaa 840 - - caagtcgctg
tgtggtttac tctttttaaa aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 900 - -
aaaaaaaaaa a - # - # - # 911 - - - - <210> SEQ ID NO 10
<211> LENGTH: 235 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 10 - - Met Ser Ser Pro Pro Pro Val
Lys Leu Ile Gl - #y Phe Phe Gly Ser Pro 1 5 - # 10 - # 15 - - Tyr
Ala Phe Arg Ala Glu Ala Ala Leu Cys Le - #u Lys Gly Val Pro Tyr 20
- # 25 - # 30 - - Glu Leu Ile Leu Glu Asp Leu Phe Gly Ser Ly - #s
Ser Glu Leu Leu Leu 35 - # 40 - # 45 - - His His Asn Pro Val His
Lys Lys Val Pro Va - #l Leu Leu His Gly Asp 50 - # 55 - # 60 - -
Gly Arg Ala Ile Ser Glu Ser Leu Val Ile Al - #a Glu Tyr Val Asp Glu
65 - # 70 - # 75 - # 80 - - Ala Phe Asp Gly Pro Pro Leu Leu Pro Ala
As - #p Pro Tyr Ala Arg Ala 85 - # 90 - # 95 - - Ala Ala Arg Phe
Trp Ala Asp Phe Ile Glu Th - #r Arg Leu Thr Lys Pro 100 - # 105 - #
110 - - Phe Phe Met Ala Ile Trp Val Glu Glu Arg As - #p Ala Arg Leu
Arg Phe 115 - # 120 - # 125 - - Glu Glu Glu Ala Lys Glu Leu Val Ala
Leu Le - #u Glu Ala Gln Leu Glu 130 - # 135 - # 140 - - Gly Lys Arg
Phe Phe Ala Gly Asp Arg Pro Gl - #y Tyr Leu Asp Val Ala 145 1 - #50
1 - #55 1 - #60 - - Ala Ser Ala Leu Gly Pro Trp Arg Ser Val Il - #e
Glu Glu Leu Asn Gly 165 - # 170 - # 175 - - Val Ala Leu Leu Ser Glu
Asp Asp His Pro As - #n Leu Cys Arg Trp Thr 180 - # 185 - # 190 - -
Arg Asp Tyr Cys Ala Phe Glu Ala Leu Lys Pr - #o Cys Met Pro Asp Arg
195 - # 200 - # 205 - - Glu Lys Leu Leu Ala Tyr Phe Thr Lys Asn Ph
- #e Asp Arg Tyr Lys Ala 210 - # 215 - # 220 - - Ala Val Asn Ala
Thr Leu Ser Gln Ser Gln Gl - #n 225 2 - #30 2 - #35 - - - -
<210> SEQ ID NO 11 <211> LENGTH: 948 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 11 - -
agcgcatgca ggtagcaatg gcgggggaga cgaagaaggg cctggtgctg ct -
#ggacttct 60 - - gggtgagccc gttcgggcag cgctgccgca tcgcgctggc
ggagaagggc at - #cgcctacg 120 - - agtactcgga gcaggagctg ctgggcggcg
ccaagagcga catcctcctc cg - #ctccaacc 180 - - cggtgcacaa gaagatcccc
gtgctcctcc acgacggccg ccccgtctgc ga - #gtccctcg 240 - - tcatcctcga
gtacctcgag gaggccttcc cggaggcctc ccccaggctg ct - #ccccgacg 300 - -
ccgcctacgc gcgcgcgcag gcccgcttct gggcggccta ctccgacaag gt -
#ctacaagg 360 - - ccggcacgcg gctgtggaag ctcaggggcg acgcgcgggc
gcaggcgcgc gc - #cgagatcg 420 - - tgcaggtggt ccggaacctc gacggcgagc
taggggacaa ggccttcttc gg - #cggcgagg 480 - - cgttcgggtt cgtggacgtg
gcgctcgtgc ccttcgtgcc gtggctcccc ag - #ctacgagc 540 - - ggtacgggga
cttcagcgtg gcggagatcg cgcccaggct ggcggcgtgg gc - #gcgccggt 600 - -
gcgcgcagcg ggagagcgtg gccaggaccc ttcacccgcc ggaaaaggtg ga -
#cgagttca 660 - - tcaacctgct caagaagacc tacggcatcg agtagtagag
cggactacta ct - #agcagagg 720 - - agatggtacc ggccgtacgt acgtggctgc
catgcagttt ttgtttcggt tt - #gtttaaac 780 - - gggactccat gaatggatgg
aactcttctt gggctccgtg tgctacatac ac - #atctgtaa 840 - - aggtgaacta
aaatcacggt aaaaactcgg aaattagttt gtaaagggtc ca - #gcccccct 900 - -
cctttataaa tagagaggta tacggctgat aaaaaaaaaa aaaaaaaa - # 948 - - -
- <210> SEQ ID NO 12 <211> LENGTH: 225 <212>
TYPE: PRT <213> ORGANISM: maize - - <400> SEQUENCE: 12
- - Met Ala Gly Glu Thr Lys Lys Gly Leu Val Le - #u Leu Asp Phe Trp
Val 1 5 - # 10 - # 15 - - Ser Pro Phe Gly Gln Arg Cys Arg Ile Ala
Le - #u Ala Glu Lys Gly Ile 20 - # 25 - # 30 - - Ala Tyr Glu Tyr
Ser Glu Gln Glu Leu Leu Gl - #y Gly Ala Lys Ser Asp 35 - # 40 - #
45 - - Ile Leu Leu Arg Ser Asn Pro Val His Lys Ly - #s Ile Pro Val
Leu Leu 50 - # 55 - # 60 - - His Asp Gly Arg Pro Val Cys Glu Ser
Leu Va - #l Ile Leu Glu Tyr Leu 65 - # 70 - # 75 - # 80 - - Glu Glu
Ala Phe Pro Glu Ala Ser Pro Arg Le - #u Leu Pro Asp Ala Ala 85 - #
90 - # 95 - - Tyr Ala Arg Ala Gln Ala Arg Phe Trp Ala Al - #a Tyr
Ser Asp Lys Val 100 - # 105 - # 110 - - Tyr Lys Ala Gly Thr Arg Leu
Trp Lys Leu Ar - #g Gly Asp Ala Arg Ala 115 - # 120 - # 125 - - Gln
Ala Arg Ala Glu Ile Val Gln Val Val Ar - #g Asn Leu Asp Gly Glu 130
- # 135 - # 140 - - Leu Gly Asp Lys Ala Phe Phe Gly Gly Glu Al - #a
Phe Gly Phe Val Asp 145 1 - #50 1 - #55 1 - #60 - - Val Ala Leu Val
Pro Phe Val Pro Trp Leu Pr - #o Ser Tyr Glu Arg Tyr 165 - # 170 - #
175 - - Gly Asp Phe Ser Val Ala Glu Ile Ala Pro Ar - #g Leu Ala Ala
Trp Ala 180 - # 185 - # 190 - - Arg Arg Cys Ala Gln Arg Glu Ser Val
Ala Ar - #g Thr Leu His Pro Pro 195 - # 200 - # 205 - - Glu Lys Val
Asp Glu Phe Ile Asn Leu Leu Ly - #s Lys Thr Tyr Gly Ile 210 - # 215
- # 220 - - Glu 225 - - - - <210> SEQ ID NO 13 <211>
LENGTH: 840 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 13 - - gttggggatg tgggcgagcc ctatggtgat
cagggtggag tgggcgctgc gg - #ctgaaggg 60 - - cgtcgagtac gagtacgtcg
acgaggacct cgccaacaag agcgccgacc tg - #ctccgcca 120 - - caacccggtg
accaagaagg tgcccgtgct cgtccacgac ggcaagccgg tc - #gcggagtc 180 - -
caccatcatc gtcgagtaca tcgacgaggt ctggaagggc ggctacccca tc -
#atgccggg 240 - - cgacccctac gagcgtgccc aggcaaggtt ctgggccagg
ttcgctgaag ac - #aagtgcaa 300 - - cgctgctctg tacccgatct tcaccgcgac
cggcgaggcg cagcgcaagg cg - #gtgcacga 360 - - ggcccagcag tgcctcaaga
ccctggagac ggccttggac gggaagaagt tc - #ttcggcgg 420 - - ggacgccgtg
ggctacctcg acatcgtcgt cgggtggttc gcgcactggc tc - #cccgtcat 480 - -
cgaggaggtg accggcgcca gcgtcgtcac cgacgaggag ctgccgctga tg -
#aaggcctg 540 - - gttcggccgg ttcctcgccg ttgacgtggt gaaggcggcc
ctgcccgaca gg - #gacaggct 600 - - cctcgccgcc aacaaggccc gccgtgagca
gctcctctcc gcgtagatgg ct - #agtaattc 660 - - tggagcagct agtttcaccg
ccgacgctca tatattgctg aataaggact gg - #ttgcactt 720 - - ttgcacgttg
tgcagtgcag ccgaggtttg gatgacctct gcccctctgt tc - #catttcag 780 - -
aatggtagtc ccataataat gcatatacat catgcataaa aaaaaaaaaa aa -
#aaaaaaaa 840 - - - - <210> SEQ ID NO 14 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 14 - - Met Trp Ala Ser Pro Met Val Ile Arg
Val Gl - #u Trp Ala Leu Arg Leu 1 5 - # 10 - # 15 - - Lys Gly Val
Glu Tyr Glu Tyr Val Asp Glu As - #p Leu Ala Asn Lys Ser 20 - # 25 -
# 30 - - Ala Asp Leu Leu Arg His Asn Pro Val Thr Ly - #s Lys Val
Pro Val Leu 35 - # 40 - # 45 - - Val His Asp Gly Lys Pro Val Ala
Glu Ser Th - #r Ile Ile Val Glu Tyr
50 - # 55 - # 60 - - Ile Asp Glu Val Trp Lys Gly Gly Tyr Pro Il -
#e Met Pro Gly Asp Pro 65 - # 70 - # 75 - # 80 - - Tyr Glu Arg Ala
Gln Ala Arg Phe Trp Ala Ar - #g Phe Ala Glu Asp Lys 85 - # 90 - #
95 - - Cys Asn Ala Ala Leu Tyr Pro Ile Phe Thr Al - #a Thr Gly Glu
Ala Gln 100 - # 105 - # 110 - - Arg Lys Ala Val His Glu Ala Gln Gln
Cys Le - #u Lys Thr Leu Glu Thr 115 - # 120 - # 125 - - Ala Leu Asp
Gly Lys Lys Phe Phe Gly Gly As - #p Ala Val Gly Tyr Leu 130 - # 135
- # 140 - - Asp Ile Val Val Gly Trp Phe Ala His Trp Le - #u Pro Val
Ile Glu Glu 145 1 - #50 1 - #55 1 - #60 - - Val Thr Gly Ala Ser Val
Val Thr Asp Glu Gl - #u Leu Pro Leu Met Lys 165 - # 170 - # 175 - -
Ala Trp Phe Gly Arg Phe Leu Ala Val Asp Va - #l Val Lys Ala Ala Leu
180 - # 185 - # 190 - - Pro Asp Arg Asp Arg Leu Leu Ala Ala Asn Ly
- #s Ala Arg Arg Glu Gln 195 - # 200 - # 205 - - Leu Leu Ser Ala
210 - - - - <210> SEQ ID NO 15 <211> LENGTH: 861
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 15 - - cggaggcgca gagcttcgac gcgcccagcg ccgagatggt
ctacagcctc gc - #cttcctgc 60 - - cgcccaccct gcccaagcag aacgacaacg
gcaacggcgg cgcgttcaac gc - #cagggacg 120 - - ccaccgtagg cagcaacgcc
gacgcgtcca gcggcaagcg cggtgtggcc gg - #gtcacagc 180 - - cggcggcgag
ccagaccaag gtgagcgcgc agaaggagga ggagatgctg aa - #gctgttcg 240 - -
agcagaggaa gaaggacctg gagaagctgc tggacatcta cgagcagcgc ct -
#ggaggagg 300 - - ccacgttcct ggccggcgac aacttcacca tcgccgacct
gtcgcacctg cc - #ctacgcgg 360 - - accacctcgt ctccgacccg cgctcccgcc
gcatgttcga gtcccgcaag aa - #cgtcagca 420 - - ggtggtggca cgacgtctcc
ggccgcgaca cctggaagta cgtcaagacc ct - #gcagcgcc 480 - - cgccgtccac
gtccaccgac gccagcgcca agaacggcca gctgggccag ca - #gcagcacc 540 - -
tgccgtcgtc caccgacggc cacggcgtga agacccaacg gctggtccag aa -
#cgagcggc 600 - - acttctagct gttgccgtcc cttcccgccg acgaataaac
tacctgcgcc gc - #cgccaccg 660 - - ccgccatcca tcaacatggt tccttgtgct
gttcgtgtcg ttttcatacg tc - #atacgtgt 720 - - cttgctgctt ttgaagctcc
gttcccgggt gcagggacct acgagtccat tc - #cgtcgttt 780 - - gctgattctg
ttcgtcgtgt aataaaatga aaaccccacc ccgttttgaa tg - #aaaaaaaa 840 - -
aaaaaaaaaa aaaaaaaaaa a - # - # 861 - - - - <210> SEQ ID NO
16 <211> LENGTH: 190 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 16 - - Met Val Tyr Ser
Leu Ala Phe Leu Pro Pro Th - #r Leu Pro Lys Gln Asn 1 5 - # 10 - #
15 - - Asp Asn Gly Asn Gly Gly Ala Phe Asn Ala Ar - #g Asp Ala Thr
Val Gly 20 - # 25 - # 30 - - Ser Asn Ala Asp Ala Ser Ser Gly Lys
Arg Gl - #y Val Ala Gly Ser Gln 35 - # 40 - # 45 - - Pro Ala Ala
Ser Gln Thr Lys Val Ser Ala Gl - #n Lys Glu Glu Glu Met 50 - # 55 -
# 60 - - Leu Lys Leu Phe Glu Gln Arg Lys Lys Asp Le - #u Glu Lys
Leu Leu Asp 65 - # 70 - # 75 - # 80 - - Ile Tyr Glu Gln Arg Leu Glu
Glu Ala Thr Ph - #e Leu Ala Gly Asp Asn 85 - # 90 - # 95 - - Phe
Thr Ile Ala Asp Leu Ser His Leu Pro Ty - #r Ala Asp His Leu Val 100
- # 105 - # 110 - - Ser Asp Pro Arg Ser Arg Arg Met Phe Glu Se - #r
Arg Lys Asn Val Ser 115 - # 120 - # 125 - - Arg Trp Trp His Asp Val
Ser Gly Arg Asp Th - #r Trp Lys Tyr Val Lys 130 - # 135 - # 140 - -
Thr Leu Gln Arg Pro Pro Ser Thr Ser Thr As - #p Ala Ser Ala Lys Asn
145 1 - #50 1 - #55 1 - #60 - - Gly Gln Leu Gly Gln Gln Gln His Leu
Pro Se - #r Ser Thr Asp Gly His 165 - # 170 - # 175 - - Gly Val Lys
Thr Gln Arg Leu Val Gln Asn Gl - #u Arg His Phe 180 - # 185 - # 190
- - - - <210> SEQ ID NO 17 <211> LENGTH: 917
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 17 - - atggcggagg tggaggcgac ggtggggcga ctgatgctgt
actcgtactg gc - #gcagctcg 60 - - tgctcccacc gtgcccgcat cgctctcaat
ctcaaaggtg tggattacga gt - #acaaggcg 120 - - gtgaaccttc tcaagggcga
gcagtctgat ccagaattcg tcaagcttaa tc - #ctatgaag 180 - - ttcgtccctg
cgttggttga tggcagttct gtaataggtg actcttacgc ga - #taacactg 240 - -
tatttggagg acaagtaccc agagcctcct cttctacctc aagaccttca aa -
#agaaagct 300 - - ttgaatcacc agattgcaag cattgtagct tctggtattc
aacctctcca ta - #acctcaca 360 - - gtgttgaggt tcattgacca gaaggttggt
gcaggggaga gtgtgttgtg ga - #ctcaacaa 420 - - caaatcgaga gaggtttcac
agctattgag aacctgatac aactaaaagg at - #gcgccggg 480 - - aagtatgcaa
caggagatga agtccaactg gcagatgtat tccttgcacc cc - #agatctat 540 - -
gcagccattg aacgcactaa aattgacatg tcaaactacc tcactcttgc ta -
#ggctccac 600 - - tcggagtaca tgtcacaccc tgcgtttgaa gcagcgctcc
ctggcaagca ac - #cggacgcc 660 - - ccttcatcct cctaggaact gcaccctagt
gtgttgttcc tctgaatata ta - #tatatata 720 - - tatgtatact tctgtaagaa
ttaataatta cagagtttcg tctgctatgt cg - #aaaaatgt 780 - - caaaagtttt
tgtgatttca gagactagcg gcatgaagcg tcgttgtgga tc - #tggccgtc 840 - -
gtcctcatgt ggcatctgtg atttcagggc atgcacttcg tcttagaagg ga -
#aaaaaaaa 900 - - aaaaaaaaaa aaaaaaa - # - # - # 917 - - - -
<210> SEQ ID NO 18 <211> LENGTH: 224 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 18 - -
Met Ala Glu Val Glu Ala Thr Val Gly Arg Le - #u Met Leu Tyr Ser Tyr
1 5 - # 10 - # 15 - - Trp Arg Ser Ser Cys Ser His Arg Ala Arg Il -
#e Ala Leu Asn Leu Lys 20 - # 25 - # 30 - - Gly Val Asp Tyr Glu Tyr
Lys Ala Val Asn Le - #u Leu Lys Gly Glu Gln 35 - # 40 - # 45 - -
Ser Asp Pro Glu Phe Val Lys Leu Asn Pro Me - #t Lys Phe Val Pro Ala
50 - # 55 - # 60 - - Leu Val Asp Gly Ser Ser Val Ile Gly Asp Se -
#r Tyr Ala Ile Thr Leu 65 - # 70 - # 75 - # 80 - - Tyr Leu Glu Asp
Lys Tyr Pro Glu Pro Pro Le - #u Leu Pro Gln Asp Leu 85 - # 90 - #
95 - - Gln Lys Lys Ala Leu Asn His Gln Ile Ala Se - #r Ile Val Ala
Ser Gly 100 - # 105 - # 110 - - Ile Gln Pro Leu His Asn Leu Thr Val
Leu Ar - #g Phe Ile Asp Gln Lys 115 - # 120 - # 125 - - Val Gly Ala
Gly Glu Ser Val Leu Trp Thr Gl - #n Gln Gln Ile Glu Arg 130 - # 135
- # 140 - - Gly Phe Thr Ala Ile Glu Asn Leu Ile Gln Le - #u Lys Gly
Cys Ala Gly 145 1 - #50 1 - #55 1 - #60 - - Lys Tyr Ala Thr Gly Asp
Glu Val Gln Leu Al - #a Asp Val Phe Leu Ala 165 - # 170 - # 175 - -
Pro Gln Ile Tyr Ala Ala Ile Glu Arg Thr Ly - #s Ile Asp Met Ser Asn
180 - # 185 - # 190 - - Tyr Leu Thr Leu Ala Arg Leu His Ser Glu Ty
- #r Met Ser His Pro Ala 195 - # 200 - # 205 - - Phe Glu Ala Ala
Leu Pro Gly Lys Gln Pro As - #p Ala Pro Ser Ser Ser 210 - # 215 - #
220 - - - - <210> SEQ ID NO 19 <211> LENGTH: 919
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 19 - - cacctgctgt atctcattac catctgcatc tggttgcccg
ttgattgaga ag - #gaggagct 60 - - gagggccatg gcgaccgaga agcccatcct
gtacaacgcc tggatcagct cc - #tgctccca 120 - - ccgtgttcgc atcgcactca
acctcaaagg tgtggattac gagtacaagt cg - #gtaaaccc 180 - - taggacagat
ccagattatg aaaaaatcaa tccaatcaaa tatattccag ca - #ttagtaga 240 - -
tggggacata gtcgtttctg attctcttgc catctcattg tatttggaag at -
#aagtatcc 300 - - tgagcatcca ctcctgccta aagatctcaa gaggaaagct
cttaatcttc ag - #attgcaaa 360 - - cattgtttgt tcaagcattc aacctcttca
aggctatgct gttattggtc tg - #cacgaggg 420 - - taggatgagc ccagatgagg
gccttcatat tgttcaaagt tatattgaca ag - #ggattcag 480 - - agcgatcgaa
aagctgttgg aaggatgtga gagtaaatat gctactggag at - #gatgtcca 540 - -
attggcagat gtgttccttg aaccacagat acatgccggc ataaatcgct tc -
#caaatcga 600 - - tatgtcgatg tacccaatct tggagaggct ccatgatgca
tacatgcaaa tt - #cccgcatt 660 - - ccaagccgcg cttcctaaaa atcaaccaga
cgcaccttca tcataatcat ca - #agattatc 720 - - tcaataattt gcatgtcatt
ttgtaataat ttggataggg agccactgct tc - #ctccatcc 780 - - cgttgtggat
caaaagggtg aacgattggc acttacctgc atggtccaat ac - #ctattata 840 - -
tttcttaaac agatactatt tacggctatt gtaatttaag cccaaaaaaa aa -
#aaaaaaaa 900 - - aaaaaaaaaa aaaaaaaaa - # - # - #919 - - - -
<210> SEQ ID NO 20 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 20 - -
Met Ala Thr Glu Lys Pro Ile Leu Tyr Asn Al - #a Trp Ile Ser Ser Cys
1 5 - # 10 - # 15 - - Ser His Arg Val Arg Ile Ala Leu Asn Leu Ly -
#s Gly Val Asp Tyr Glu 20 - # 25 - # 30 - - Tyr Lys Ser Val Asn Pro
Arg Thr Asp Pro As - #p Tyr Glu Lys Ile Asn 35 - # 40 - # 45 - -
Pro Ile Lys Tyr Ile Pro Ala Leu Val Asp Gl - #y Asp Ile Val Val Ser
50 - # 55 - # 60 - - Asp Ser Leu Ala Ile Ser Leu Tyr Leu Glu As -
#p Lys Tyr Pro Glu His 65 - # 70 - # 75 - # 80 - - Pro Leu Leu Pro
Lys Asp Leu Lys Arg Lys Al - #a Leu Asn Leu Gln Ile 85 - # 90 - #
95 - - Ala Asn Ile Val Cys Ser Ser Ile Gln Pro Le - #u Gln Gly Tyr
Ala Val 100 - # 105 - # 110 - - Ile Gly Leu His Glu Gly Arg Met Ser
Pro As - #p Glu Gly Leu His Ile 115 - # 120 - # 125 - - Val Gln Ser
Tyr Ile Asp Lys Gly Phe Arg Al - #a Ile Glu Lys Leu Leu 130 - # 135
- # 140 - - Glu Gly Cys Glu Ser Lys Tyr Ala Thr Gly As - #p Asp Val
Gln Leu Ala 145 1 - #50 1 - #55 1 - #60 - - Asp Val Phe Leu Glu Pro
Gln Ile His Ala Gl - #y Ile Asn Arg Phe Gln 165 - # 170 - # 175 - -
Ile Asp Met Ser Met Tyr Pro Ile Leu Glu Ar - #g Leu His Asp Ala Tyr
180 - # 185 - # 190 - - Met Gln Ile Pro Ala Phe Gln Ala Ala Leu Pr
- #o Lys Asn Gln Pro Asp 195 - # 200 - # 205 - - Ala Pro Ser Ser
210 - - - - <210> SEQ ID NO 21 <211> LENGTH: 996
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 21 - - catcgatccg ccattgctca ccgcacaagt gcacgctcac
ctcacacacg ca - #gctaagta 60 - - gctaacgccg taggcgagaa caagaaaagg
ctcgacatgg ccgaggagaa ga - #agcagggc 120 - - ctgcagctgc tggacttctg
ggtgagccca ttcgggcagc gctgccgcat cg - #cgctggac 180 - - gagaagggcc
tggcctacga gtacctggag caggacctga ggaacaagag cg - #agctgctc 240 - -
ctccgcgcca acccggtgca caagaagatc cccgtgctgc tgcacgacgg cc -
#gccccgtc 300 - - tgcgagtccc tcgtcatcgt gcagtacctc gacgaggcgt
tcccggaggc gg - #cgccggcg 360 - - ctgctccccg ccgaccccta cgcgcgcgcg
caggcccgct tctgggcgga ct -
#acgtcgac 420 - - aagaagctgt acgactgcgg cacccggctg tggaagctca
agggggacgg cc - #aggcgcag 480 - - gcgcgcgccg agatggtcga gatcctccgc
acgctggagg gcgcgctcgg cg - #acgggccc 540 - - ttcttcggtg gcgacgccct
cggcttcgtc gacgtcgcgc tcgtgccctt ca - #cgtcctgg 600 - - ttcctcgcct
acgaccgctt cggcggcgtc agcgtggaga aggagtgccc ga - #ggctggcc 660 - -
gcctgggcca agcgctgcgc cgagcgcccc agcgtcgcca agaacctcta cc -
#cgcccgag 720 - - aaggtctacg acttcgtctg cgggatgaag aagaggctgg
gcatcgagta ga - #gcatccat 780 - - cggtcggccg gtggctggcc gggagtaata
atgacgaacc aataatctag tt - #ttggtttt 840 - - agtgtgctca gcagagcagt
tcgtgttcat gagttcgtcg tcgttgtatt tt - #ctattgtc 900 - - agcggtggca
gcgccgtacg tgttgcctcg tacaccacaa ccgaataaat gg - #ttatgaat 960 - -
ttcttcttgt tgtcttaaaa aaaaaaaaaa aaaaaa - # - # 996 - - - -
<210> SEQ ID NO 22 <211> LENGTH: 224 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 22 - -
Met Ala Glu Glu Lys Lys Gln Gly Leu Gln Le - #u Leu Asp Phe Trp Val
1 5 - # 10 - # 15 - - Ser Pro Phe Gly Gln Arg Cys Arg Ile Ala Le -
#u Asp Glu Lys Gly Leu 20 - # 25 - # 30 - - Ala Tyr Glu Tyr Leu Glu
Gln Asp Leu Arg As - #n Lys Ser Glu Leu Leu 35 - # 40 - # 45 - -
Leu Arg Ala Asn Pro Val His Lys Lys Ile Pr - #o Val Leu Leu His Asp
50 - # 55 - # 60 - - Gly Arg Pro Val Cys Glu Ser Leu Val Ile Va -
#l Gln Tyr Leu Asp Glu 65 - # 70 - # 75 - # 80 - - Ala Phe Pro Glu
Ala Ala Pro Ala Leu Leu Pr - #o Ala Asp Pro Tyr Ala 85 - # 90 - #
95 - - Arg Ala Gln Ala Arg Phe Trp Ala Asp Tyr Va - #l Asp Lys Lys
Leu Tyr 100 - # 105 - # 110 - - Asp Cys Gly Thr Arg Leu Trp Lys Leu
Lys Gl - #y Asp Gly Gln Ala Gln 115 - # 120 - # 125 - - Ala Arg Ala
Glu Met Val Glu Ile Leu Arg Th - #r Leu Glu Gly Ala Leu 130 - # 135
- # 140 - - Gly Asp Gly Pro Phe Phe Gly Gly Asp Ala Le - #u Gly Phe
Val Asp Val 145 1 - #50 1 - #55 1 - #60 - - Ala Leu Val Pro Phe Thr
Ser Trp Phe Leu Al - #a Tyr Asp Arg Phe Gly 165 - # 170 - # 175 - -
Gly Val Ser Val Glu Lys Glu Cys Pro Arg Le - #u Ala Ala Trp Ala Lys
180 - # 185 - # 190 - - Arg Cys Ala Glu Arg Pro Ser Val Ala Lys As
- #n Leu Tyr Pro Pro Glu 195 - # 200 - # 205 - - Lys Val Tyr Asp
Phe Val Cys Gly Met Lys Ly - #s Arg Leu Gly Ile Glu 210 - # 215 - #
220 - - - - <210> SEQ ID NO 23 <211> LENGTH: 895
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 23 - - ggcacgagac gacatcgaag gagcctgcga agcgagcgag
agtctataat gg - #cggacgga 60 - - ggcgagctgc agctgctggg ctcatggtac
agcccctacg tgatccgcgc ca - #aggtggcg 120 - - ctggggctga aggggctcag
ctacgagttc gtcgaggagg acctctcccg ca - #agagcgac 180 - - ctgctgctga
agctcaaccc ggtgcacagg aaggtgcccg tgctggtcca cg - #gcggccgc 240 - -
cccgtgtgcg agtcgctcgt catcctgcag tacgtcgacg agacctgggc ag -
#gcaccggg 300 - - acccctctcc tccccgccga cgcctacgac cgcgccatgg
ctcgcttctg gg - #cagcctac 360 - - gtcgacgaca agttctacaa ggagtggaac
cggctgttct ggtcgacgac gg - #cggagaag 420 - - gcggcggagg cgctcggcgt
cgtcgtcccc gtggtggaga cgctggagca gg - #cgttcagg 480 - - gagtgctcca
aagggaaacc ttcttcggcg gcgacgccgt cgggctcgtg ga - #catcgcgc 540 - -
tcgggagctt cgtggtgtgg atcagggtgg tggacgaggc ggccggcgta aa -
#gcttctgg 600 - - acgaggccaa gttcccggcc ttgacggcgt gggcggagcg
cttcttggcg gt - #ggacgccg 660 - - tgaaggaggt gatgccggac gccggaaggc
tgttggagca ctacaagggg tt - #tctggcta 720 - - aacggtctcc acctgctggt
tactgaacgc tgtaactgta agcctgtaac ag - #caagctca 780 - - gtgttcgtgt
acttttccgt gcgttaacgt gtactagagt tcaggaaagg ct - #ttgattct 840 - -
gtccagagtc cagacgaata aacgaatgtt ttttataaaa aaaaaaaaaa aa - #aaa
895 - - - - <210> SEQ ID NO 24 <211> LENGTH: 180
<212> TYPE: PRT <213> ORGANISM: maize - - <400>
SEQUENCE: 24 - - Met Ala Asp Gly Gly Glu Leu Gln Leu Leu Gl - #y
Ser Trp Tyr Ser Pro 1 5 - # 10 - # 15 - - Tyr Val Ile Arg Ala Lys
Val Ala Leu Gly Le - #u Lys Gly Leu Ser Tyr 20 - # 25 - # 30 - -
Glu Phe Val Glu Glu Asp Leu Ser Arg Lys Se - #r Asp Leu Leu Leu Lys
35 - # 40 - # 45 - - Leu Asn Pro Val His Arg Lys Val Pro Val Le -
#u Val His Gly Gly Arg 50 - # 55 - # 60 - - Pro Val Cys Glu Ser Leu
Val Ile Leu Gln Ty - #r Val Asp Glu Thr Trp 65 - # 70 - # 75 - # 80
- - Ala Gly Thr Gly Thr Pro Leu Leu Pro Ala As - #p Ala Tyr Asp Arg
Ala 85 - # 90 - # 95 - - Met Ala Arg Phe Trp Ala Ala Tyr Val Asp As
- #p Lys Phe Tyr Lys Glu 100 - # 105 - # 110 - - Trp Asn Arg Leu
Phe Trp Ser Thr Thr Ala Gl - #u Lys Ala Ala Glu Ala 115 - # 120 - #
125 - - Leu Gly Val Val Val Pro Val Val Glu Thr Le - #u Glu Gln Ala
Phe Arg 130 - # 135 - # 140 - - Glu Cys Ser Lys Gly Lys Pro Ser Ser
Ala Al - #a Thr Pro Ser Gly Ser 145 1 - #50 1 - #55 1 - #60 - - Trp
Thr Ser Arg Ser Gly Ala Ser Trp Cys Gl - #y Ser Gly Trp Trp Thr 165
- # 170 - # 175 - - Arg Arg Pro Ala 180 - - - - <210> SEQ ID
NO 25 <211> LENGTH: 1279 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 25 - - ctgcaggttc
agttcagtag tgtgctctga cagtgagatg gcgagcgtga ag - #gttttcgg 60 - -
gtcacccacc tcggcggagg tcgcccgcgt gctcatgtgc ctcttcgaga ag -
#gaggtgga 120 - - gttccagctg atccgcgtcg acgcctaccg cggcaccaag
cgcatgcccc ag - #tacctcaa 180 - - gctgcagccg caaggcgagg cgctcacctt
cgaggacgag agcctcaccc tc - #tccgactc 240 - - cagggggatc ctccgccaca
tctcccacaa gtacgcgaag cagggcaacc cg - #ttacctga 300 - - ttggcacggg
cgcgctggag cgggcgtcca tcgagcagtg gctgcagacg ga - #ggcgcaga 360 - -
gcttcgacgc gcccagcgcc gagatggtct acagcctcgc cttcctgccg cc -
#caccctgc 420 - - ccaagcagaa cgacaacggc aacggcggcg cgttcaacgc
cagggacgcc ac - #cgtaggca 480 - - gcaacgccga cgcgtccagc ggcaagcgcg
gtgtggccgg gtcacagccg gc - #ggcgagcc 540 - - agaccaaggt gagcgcgcag
aaggaggagg agatgctgaa gctgttcgag ca - #gaggaaga 600 - - aggacctgga
gaagctgctg gacatctacg agcagcgcct ggaggaggcc ac - #gttcctgg 660 - -
ccggcgacaa cttcaccatc gccgacctgt cgcacctgcc ctacgcggac ca -
#cctcgtct 720 - - ccgacccgcg ctcccgccgc atgttcgagt cccgcaagaa
cgtcagcagg tg - #gtggcacg 780 - - acgtctccgg ccgcgacacc tggaagtacg
tcaagaccct gcagcgcccg cc - #gtccacgt 840 - - ccaccgacgc cagcgccaag
aacggccagc tgggccagca gcagcacctg cc - #gtcgtcca 900 - - ccgacggcca
cggcgtgaag acccaacggc tggtccagaa cgagcggcac tt - #ctagctgt 960 - -
tgccgtccct tcccgccgac gaataaacta cctgcgccgc cgccaccgcc gc -
#catccatc 1020 - - aacatggttc cttgtgctgt tcgtgtcgtt ttcatacgtc
atacgtgtct tg - #ctgctttt 1080 - - gaagctccgt tcccgggtgc agggacctac
gagtccattc cgtcgtttgc tg - #attctgtt 1140 - - cgtcgtgtaa taaaatgaaa
accccacccc gttttgaatg aaattcaatt ct - #cagtgtcg 1200 - - tgtgaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 1260 - -
aaaaaaaaaa aaaaaaaaa - # - # 127 - #9 - - - - <210> SEQ ID NO
26 <211> LENGTH: 370 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 26 - - Met Ala Ser Val
Lys Val Phe Gly Ser Pro Th - #r Ser Ala Glu Val Ala 1 5 - # 10 - #
15 - - Arg Val Leu Met Cys Leu Phe Glu Lys Glu Va - #l Glu Phe Gln
Leu Ile 20 - # 25 - # 30 - - Arg Val Asp Ala Tyr Arg Gly Thr Lys
Arg Me - #t Pro Gln Tyr Leu Lys 35 - # 40 - # 45 - - Leu Gln Pro
Gln Gly Glu Ala Leu Thr Phe Gl - #u Asp Glu Ser Leu Thr 50 - # 55 -
# 60 - - Leu Ser Asp Ser Arg Gly Ile Leu Arg His Il - #e Ser His
Lys Tyr Ala 65 - # 70 - # 75 - # 80 - - Lys Gln Gly Asn Pro Leu Pro
Asp Trp His Gl - #y Arg Ala Gly Ala Gly 85 - # 90 - # 95 - - Val
His Arg Ala Val Ala Ala Asp Gly Gly Al - #a Glu Leu Arg Arg Ala 100
- # 105 - # 110 - - Gln Arg Arg Asp Gly Leu Gln Pro Arg Leu Pr - #o
Ala Ala His Pro Ala 115 - # 120 - # 125 - - Gln Ala Glu Arg Gln Arg
Gln Arg Arg Arg Va - #l Gln Arg Gln Gly Arg 130 - # 135 - # 140 - -
His Arg Arg Gln Gln Arg Arg Arg Val Gln Ar - #g Gln Ala Arg Cys Gly
145 1 - #50 1 - #55 1 - #60 - - Arg Val Thr Ala Gly Gly Glu Pro Asp
Gln Gl - #y Glu Arg Ala Glu Gly 165 - # 170 - # 175 - - Gly Gly Asp
Ala Glu Ala Val Arg Ala Glu Gl - #u Glu Gly Pro Gly Glu 180 - # 185
- # 190 - - Ala Ala Gly His Leu Arg Ala Ala Pro Gly Gl - #y Gly His
Val Pro Gly 195 - # 200 - # 205 - - Arg Arg Gln Leu His His Arg Arg
Pro Val Al - #a Pro Ala Leu Arg Gly 210 - # 215 - # 220 - - Pro Pro
Arg Leu Arg Pro Ala Leu Pro Pro Hi - #s Val Arg Val Pro Gln 225 2 -
#30 2 - #35 2 - #40 - - Glu Arg Gln Gln Val Val Ala Arg Arg Leu Ar
- #g Pro Arg His Leu Glu 245 - # 250 - # 255 - - Val Arg Gln Asp
Pro Ala Ala Pro Ala Val Hi - #s Val His Arg Arg Gln 260 - # 265 - #
270 - - Arg Gln Glu Arg Pro Ala Gly Pro Ala Ala Al - #a Pro Ala Val
Val His 275 - # 280 - # 285 - - Arg Arg Pro Arg Arg Glu Asp Pro Thr
Ala Gl - #y Pro Glu Arg Ala Ala 290 - # 295 - # 300 - - Leu Leu Ala
Val Ala Val Pro Ser Arg Arg Ar - #g Ile Asn Tyr Leu Arg 305 3 - #10
3 - #15 3 - #20 - - Arg Arg His Arg Arg His Pro Ser Thr Trp Ph - #e
Leu Val Leu Phe Val 325 - # 330 - # 335 - - Ser Phe Ser Tyr Val Ile
Arg Val Leu Leu Le - #u Leu Lys Leu Arg Ser 340 - # 345 - # 350 - -
Arg Val Gln Gly Pro Thr Ser Pro Phe Arg Ar - #g Leu Leu Ile Leu Phe
355 - # 360 - # 365 - - Val Val 370 - - - - <210> SEQ ID NO
27 <211> LENGTH: 1198 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 27 - - ctggctcacc
tcacctgcag caggcctcgt ctcggtcatc cagcgcattg ct - #ctcaatcg 60 - -
ctgcagcgca tccagtccaa acacacaccg gtcgaatcga gcaatggccg cg -
#gggctgca 120 - - ggtgttcgga cagccggctt ccaccgacgt cgccagggtg
ctgacctgcc tc - #ttcgagaa 180 - - gaacctcgag ttcgagctcg tccgcaccga
caccttcaag aagtcgcaca ag - #ctccccga 240 - - gttcatcaag ctgagggatc
ctaccgggca ggtgactttc aagcacggtg ac - #aagacaat 300 - - cgttgattcc
aggactatct gccggtacct gtgcacgcag ttcccggacg ac - #gggtacaa 360 - -
gaagctgtac ggcacggggt cgctggagcg ggcgtccata gagcagtggc tg -
#caggcgga 420 - - ggcgcagagc ttcgacgcgc cgagctcgga gctggcgttc
cagctggcgt tc -
#gcgccgca 480 - - cctcaaggac gtgcggcccg acgaggcccg cgtcgcggag
aacgagaaga ag - #ctgcacag 540 - - catgctgggc gtctacgacg acatcctctc
caagaacgag tacctcgccg gc - #gacgactt 600 - - cacactggcc gacctctccc
acctgccaaa ctcccactac atcgtcaact cc - #tccgacag 660 - - gggcaggaag
ctcttcaccg ccaggaagca cgtggccagg tggtacgaca ag - #atctccac 720 - -
ccgcgactcc tggaggcagg tcatgaagat gcagagggag caccccggcg cg -
#ttcgagtg 780 - - atgcgtcgtg cttccttctc tctgcatgca tgcgcgcgtc
gcggcgtgtt cc - #tcgtcgtc 840 - - gccggcttcg tggtcgtcag gcttcacacc
gtggtgtgtg gttgtcgagt tc - #gtcgtatt 900 - - tcgtatcgta tcgtatcgta
tcgtacgtac gtcctgtggg ctaaaataac gt - #ggagcctg 960 - - cgcctgccta
cggtgtctct cgtgtcttcc tttcgctgca tatataagca gt - #gtcttttt 1020 - -
ctgggtattg tatggtgacc taatcatcaa ctccttttgc aatattggcc aa -
#ttcaataa 1080 - - aaatatgtcc gggcattttg ctcgttcgca ctaaaaaaaa
aaaaaaaaaa aa - #aaaaaaaa 1140 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa - #aaaaaa 1198 - - - - <210> SEQ ID
NO 28 <211> LENGTH: 225 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 28 - - Met Ala Ala Gly
Leu Gln Val Phe Gly Gln Pr - #o Ala Ser Thr Asp Val 1 5 - # 10 - #
15 - - Ala Arg Val Leu Thr Cys Leu Phe Glu Lys As - #n Leu Glu Phe
Glu Leu 20 - # 25 - # 30 - - Val Arg Thr Asp Thr Phe Lys Lys Ser
His Ly - #s Leu Pro Glu Phe Ile 35 - # 40 - # 45 - - Lys Leu Arg
Asp Pro Thr Gly Gln Val Thr Ph - #e Lys His Gly Asp Lys 50 - # 55 -
# 60 - - Thr Ile Val Asp Ser Arg Thr Ile Cys Arg Ty - #r Leu Cys
Thr Gln Phe 65 - # 70 - # 75 - # 80 - - Pro Asp Asp Gly Tyr Lys Lys
Leu Tyr Gly Th - #r Gly Ser Leu Glu Arg 85 - # 90 - # 95 - - Ala
Ser Ile Glu Gln Trp Leu Gln Ala Glu Al - #a Gln Ser Phe Asp Ala 100
- # 105 - # 110 - - Pro Ser Ser Glu Leu Ala Phe Gln Leu Ala Ph - #e
Ala Pro His Leu Lys 115 - # 120 - # 125 - - Asp Val Arg Pro Asp Glu
Ala Arg Val Ala Gl - #u Asn Glu Lys Lys Leu 130 - # 135 - # 140 - -
His Ser Met Leu Gly Val Tyr Asp Asp Ile Le - #u Ser Lys Asn Glu Tyr
145 1 - #50 1 - #55 1 - #60 - - Leu Ala Gly Asp Asp Phe Thr Leu Ala
Asp Le - #u Ser His Leu Pro Asn 165 - # 170 - # 175 - - Ser His Tyr
Ile Val Asn Ser Ser Asp Arg Gl - #y Arg Lys Leu Phe Thr 180 - # 185
- # 190 - - Ala Arg Lys His Val Ala Arg Trp Tyr Asp Ly - #s Ile Ser
Thr Arg Asp 195 - # 200 - # 205 - - Ser Trp Arg Gln Val Met Lys Met
Gln Arg Gl - #u His Pro Gly Ala Phe 210 - # 215 - # 220 - - Glu 225
- - - - <210> SEQ ID NO 29 <211> LENGTH: 1134
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 29 - - tcatcatcca ggcgccgcag tgtaggtcta gatcatccaa
tccaacacac cg - #gtcgagca 60 - - atggcggcag ggctgcaggt gttcggacag
ccggcgtcca ccgacgtcgc ga - #gggtgctg 120 - - acctgcctct tcgagaagaa
cctcgagttc gagctcatcc gcaccgacac ct - #tcaagaag 180 - - tcccacaagc
tccccgagtt catcaagcta agggatccta ctgggcaggt ga - #ctttcaag 240 - -
cacggtgaca aaacaatcgt tgattccagg gccatttgcc ggtacctgtg ca -
#cgcagttc 300 - - ccggacgacg ggtacaagaa gctgtacggg acggggtcgc
tggagcgggc gt - #ccatagag 360 - - cagtggctgc aggcggaggc ccagagcttc
gacgcgccga gctcggagct gg - #cgttccag 420 - - ctggcgttcg cgccgcacct
caagaacgtg cggcccgacg aggcccgcgc cg - #cggagaac 480 - - gagaggaagc
tgcacggcat gctgggcgtc tacgacgaca tcctctccaa ga - #acgagtac 540 - -
ctcgccggcg acgacttcac cctggccgac ctctcccacc tgcccaactc cc -
#actacatc 600 - - gtcaactcct ccgacagggg cagaaagctc ttcaccgcca
ggaagcacgt cg - #ccaggtgg 660 - - tacgacaaga tctccacccg cgactcgtgg
aggcaggtca tcaagatgca ga - #gggagcac 720 - - cccggcgcgt tcgagtgatc
ggtcggtggt ctacgcggtg atgcatgcat gc - #atgcatgc 780 - - gccgcggcgt
gttcctcgat cgccgccagc caccggcggc ttcgtcgtcg tc - #aggcttcg 840 - -
taccttgacg gggttgtcga cttcgtcgta cgtccctgtg gcctgtgggc ta -
#aaataacg 900 - - tgaagcctgc ctacgcggtg tctcgtgtct taccttttaa
atttgcacca ta - #tatacgca 960 - - ctgtcttttc tgggtatttg ttgtattgtg
atgtacggag tattcatcaa ct - #ccttttgc 1020 - - aagattggtc aattattcag
gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 1080 - - aaaaaaaaaa
agaaaaaaaa aaaanaaaaa aaaaaaaaaa aaaaaaaaaa aa - #na 1134 - - - -
<210> SEQ ID NO 30 <211> LENGTH: 225 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 30 - -
Met Ala Ala Gly Leu Gln Val Phe Gly Gln Pr - #o Ala Ser Thr Asp Val
1 5 - # 10 - # 15 - - Ala Arg Val Leu Thr Cys Leu Phe Glu Lys As -
#n Leu Glu Phe Glu Leu 20 - # 25 - # 30 - - Ile Arg Thr Asp Thr Phe
Lys Lys Ser His Ly - #s Leu Pro Glu Phe Ile 35 - # 40 - # 45 - -
Lys Leu Arg Asp Pro Thr Gly Gln Val Thr Ph - #e Lys His Gly Asp Lys
50 - # 55 - # 60 - - Thr Ile Val Asp Ser Arg Ala Ile Cys Arg Ty -
#r Leu Cys Thr Gln Phe 65 - # 70 - # 75 - # 80 - - Pro Asp Asp Gly
Tyr Lys Lys Leu Tyr Gly Th - #r Gly Ser Leu Glu Arg 85 - # 90 - #
95 - - Ala Ser Ile Glu Gln Trp Leu Gln Ala Glu Al - #a Gln Ser Phe
Asp Ala 100 - # 105 - # 110 - - Pro Ser Ser Glu Leu Ala Phe Gln Leu
Ala Ph - #e Ala Pro His Leu Lys 115 - # 120 - # 125 - - Asn Val Arg
Pro Asp Glu Ala Arg Ala Ala Gl - #u Asn Glu Arg Lys Leu 130 - # 135
- # 140 - - His Gly Met Leu Gly Val Tyr Asp Asp Ile Le - #u Ser Lys
Asn Glu Tyr 145 1 - #50 1 - #55 1 - #60 - - Leu Ala Gly Asp Asp Phe
Thr Leu Ala Asp Le - #u Ser His Leu Pro Asn 165 - # 170 - # 175 - -
Ser His Tyr Ile Val Asn Ser Ser Asp Arg Gl - #y Arg Lys Leu Phe Thr
180 - # 185 - # 190 - - Ala Arg Lys His Val Ala Arg Trp Tyr Asp Ly
- #s Ile Ser Thr Arg Asp 195 - # 200 - # 205 - - Ser Trp Arg Gln
Val Ile Lys Met Gln Arg Gl - #u His Pro Gly Ala Phe 210 - # 215 - #
220 - - Glu 225 - - - - <210> SEQ ID NO 31 <211>
LENGTH: 1007 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 31 - - aacacaggct gttgtttgct tcttttggta
aaggctttag ctgcggcaga tc - #caccggcg 60 - - gcgccccgag aatcgaagat
gccggtgaag gtgttcggat cgccgacgtc gg - #cggaggtc 120 - - gcccgcgtcc
tggcctgcct gttcgagaag gacgtcgagt tccagctcat cc - #gcgtcgac 180 - -
tccttccgcg gcaccaagcg cctgccccag tacctcaagc tccagccgca cg -
#gcgaggcg 240 - - ctcaccttcg aggacggcaa cgtcaccctc gtcgagtcga
ggaagatcct gc - #gccacatc 300 - - gccgacaagt acaagaacca ggggtacagg
gacctgttcg gcccgggcgc gc - #tggagcgg 360 - - gcctccatcg agcagtggct
gcagacggag gcgcagagct tcgacgtccc ca - #gcgccgac 420 - - atggtctaca
gcctcgccta cctgccgccc gacatgcagc tcgacggcag gg - #gcgtcggc 480 - -
ggcctcccgg cggcgacggg gacgatgaac ccggcgcacc ggcagaaggt gg -
#aggagatg 540 - - ctgcagctgt tcgagaagag ccgcaggcag ctgggcaagc
tgctggacat ct - #acgagcag 600 - - cgccttggcg aggaggcctt cctggccgga
ggcaagttca cgctcgccga cc - #tgtcccac 660 - - ctgcccaacg ccgaccgcct
cgccggcgac ccgcggtccg cacgcctcat gg - #agtcgcgc 720 - - aggaacgtca
gcaagtggtg ggacaccgta tcccgccgcg actcttgggt ca - #gggtcaag 780 - -
gagttgcagc gcccgccgtc cgcggaggcg cccttctgat gtcgatcgat cg -
#caaattaa 840 - - ggcggtggcc tttgctcaag cctacgtgtt cggtttctgc
ataatttttt aa - #taaataaa 900 - - cgccactggc ccctctacgt cattggcgat
tgttcattgt gtaataaatc gt - #tcaagagc 960 - - atatgatgct tcttgccgtg
aaaaaaaaaa aaaaaaaaaa aaaaaaa - # 1007 - - - - <210> SEQ ID
NO 32 <211> LENGTH: 246 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 32 - - Met Pro Val Lys
Val Phe Gly Ser Pro Thr Se - #r Ala Glu Val Ala Arg 1 5 - # 10 - #
15 - - Val Leu Ala Cys Leu Phe Glu Lys Asp Val Gl - #u Phe Gln Leu
Ile Arg 20 - # 25 - # 30 - - Val Asp Ser Phe Arg Gly Thr Lys Arg
Leu Pr - #o Gln Tyr Leu Lys Leu 35 - # 40 - # 45 - - Gln Pro His
Gly Glu Ala Leu Thr Phe Glu As - #p Gly Asn Val Thr Leu 50 - # 55 -
# 60 - - Val Glu Ser Arg Lys Ile Leu Arg His Ile Al - #a Asp Lys
Tyr Lys Asn 65 - # 70 - # 75 - # 80 - - Gln Gly Tyr Arg Asp Leu Phe
Gly Pro Gly Al - #a Leu Glu Arg Ala Ser 85 - # 90 - # 95 - - Ile
Glu Gln Trp Leu Gln Thr Glu Ala Gln Se - #r Phe Asp Val Pro Ser 100
- # 105 - # 110 - - Ala Asp Met Val Tyr Ser Leu Ala Tyr Leu Pr - #o
Pro Asp Met Gln Leu 115 - # 120 - # 125 - - Asp Gly Arg Gly Val Gly
Gly Leu Pro Ala Al - #a Thr Gly Thr Met Asn 130 - # 135 - # 140 - -
Pro Ala His Arg Gln Lys Val Glu Glu Met Le - #u Gln Leu Phe Glu Lys
145 1 - #50 1 - #55 1 - #60 - - Ser Arg Arg Gln Leu Gly Lys Leu Leu
Asp Il - #e Tyr Glu Gln Arg Leu 165 - # 170 - # 175 - - Gly Glu Glu
Ala Phe Leu Ala Gly Gly Lys Ph - #e Thr Leu Ala Asp Leu 180 - # 185
- # 190 - - Ser His Leu Pro Asn Ala Asp Arg Leu Ala Gl - #y Asp Pro
Arg Ser Ala 195 - # 200 - # 205 - - Arg Leu Met Glu Ser Arg Arg Asn
Val Ser Ly - #s Trp Trp Asp Thr Val 210 - # 215 - # 220 - - Ser Arg
Arg Asp Ser Trp Val Arg Val Lys Gl - #u Leu Gln Arg Pro Pro 225 2 -
#30 2 - #35 2 - #40 - - Ser Ala Glu Ala Pro Phe 245 - - - -
<210> SEQ ID NO 33 <211> LENGTH: 911 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 33 - -
cggagcaaga ggaaagccat ggctacgccg gcggcggtga tgaagttgta cg -
#ggtgggct 60 - - atctcgccgt tcgtgtcgcg ggctctgctg gccctggagg
aggccggcgt cg - #actacgag 120 - - ctcgtcccca tgagccccca ggccggcgac
caccggcgcc cggagcacct cg - #ccaggaac 180 - - cctttcgcca tggtgccggt
gctcgaggac ggcgacctca cgctctttga at - #cccgggcg 240 - - atcgcgaggc
acgttctccg caagcacagg ccggagctcc tgggcgccgg cg - #ccggcggc 300 - -
agcctcgagc gggcggcgat ggtggacgtg tggctcgagg tggaggcgca cc -
#agctgagc 360 - - ccgccagcgg tcgccatcgt ggtggagtgc ttcgctgcgc
cgctgctcgg cc - #gcgagcgc 420 - - gaccagacgg tcgtcgacga gaacgtggag
aagctcagga aggtgctcga gg - #tgtacgag 480 - - gcgcggcttg gcgagtgcag
gtacctcgcc ggcgacttcc tcagcctcgc cg - #acctcagc 540 - - cccttcacca
tcatgcactg catcatggcc accgagtacg ccgccgccct gg - #tcgaggcg 600 - -
ctcccgcgcg tcagcgcctg gtgggagggc ctcgccgcgc gccccgcggc ca -
#agaaggtg 660 - - gcggagttca taccggtcgg cgcggccgga ctgctggagc
accctcccaa ac - #aacaggat 720
- - tgatgcatga tgaagcaagc ctgcctaatg tgcctgttgc gcttaatact tt -
#cccacgtg 780 - - tactttccca caacgttgac agaagttatt caataactag
tctctatgta ac - #gtaatggt 840 - - gtggtgtgca cttcaatgaa taccatgagg
tggctggttc aaaaaaaaaa aa - #aaaaaaaa 900 - - aaaaaaaaaa a - # - # -
# 911 - - - - <210> SEQ ID NO 34 <211> LENGTH: 234
<212> TYPE: PRT <213> ORGANISM: maize - - <400>
SEQUENCE: 34 - - Met Ala Thr Pro Ala Ala Val Met Lys Leu Ty - #r
Gly Trp Ala Ile Ser 1 5 - # 10 - # 15 - - Pro Phe Val Ser Arg Ala
Leu Leu Ala Leu Gl - #u Glu Ala Gly Val Asp 20 - # 25 - # 30 - -
Tyr Glu Leu Val Pro Met Ser Pro Gln Ala Gl - #y Asp His Arg Arg Pro
35 - # 40 - # 45 - - Glu His Leu Ala Arg Asn Pro Phe Ala Met Va -
#l Pro Val Leu Glu Asp 50 - # 55 - # 60 - - Gly Asp Leu Thr Leu Phe
Glu Ser Arg Ala Il - #e Ala Arg His Val Leu 65 - # 70 - # 75 - # 80
- - Arg Lys His Arg Pro Glu Leu Leu Gly Ala Gl - #y Ala Gly Gly Ser
Leu 85 - # 90 - # 95 - - Glu Arg Ala Ala Met Val Asp Val Trp Leu Gl
- #u Val Glu Ala His Gln 100 - # 105 - # 110 - - Leu Ser Pro Pro
Ala Val Ala Ile Val Val Gl - #u Cys Phe Ala Ala Pro 115 - # 120 - #
125 - - Leu Leu Gly Arg Glu Arg Asp Gln Thr Val Va - #l Asp Glu Asn
Val Glu 130 - # 135 - # 140 - - Lys Leu Arg Lys Val Leu Glu Val Tyr
Glu Al - #a Arg Leu Gly Glu Cys 145 1 - #50 1 - #55 1 - #60 - - Arg
Tyr Leu Ala Gly Asp Phe Leu Ser Leu Al - #a Asp Leu Ser Pro Phe 165
- # 170 - # 175 - - Thr Ile Met His Cys Ile Met Ala Thr Glu Ty - #r
Ala Ala Ala Leu Val 180 - # 185 - # 190 - - Glu Ala Leu Pro Arg Val
Ser Ala Trp Trp Gl - #u Gly Leu Ala Ala Arg 195 - # 200 - # 205 - -
Pro Ala Ala Lys Lys Val Ala Glu Phe Ile Pr - #o Val Gly Ala Ala Gly
210 - # 215 - # 220 - - Leu Leu Glu His Pro Pro Lys Gln Gln Asp 225
2 - #30 - - - - <210> SEQ ID NO 35 <211> LENGTH: 1098
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 35 - - gcagcattgt accctatgtt catgccacca tggaagggct
tcgtattgga gc - #accgatta 60 - - tgcaggttta tcatgagaaa tcttttatct
tacctgatgt ttcaagggtg ct - #tgcttgcc 120 - - tttatgagaa ggatgtcaag
tttgagactc acacagcctc atacaggagc ct - #actcggat 180 - - tgcaggcatc
atctcatgct ccagttccat tctatgaagg ccctactttt ct - #agaagaat 240 - -
ccagagaaat ctgccgttat atagcagaaa agtatgaaaa tcaaggatat cc -
#gttcctcc 300 - - ttggaaagga tgcccttgag agggcttcaa ttgaacaatg
gctccacaac ga - #ggagcatg 360 - - ctttcaaccc tccgagccgg gccttgttct
ttcatttggc ctttcccctg gg - #tgaaggag 420 - - aagatgatga tattgatgtt
catacaagga agctagaaga ggttctggaa gt - #ttatgagc 480 - - aaaggctcag
tgacagcgaa ttccttgttg gaaacaagtt cactcttgcc ga - #ccttgttc 540 - -
acctgccaaa ttcccactat atcaaagcat ctaacaagtt tctttacctt ta -
#tgattcga 600 - - ggaaaaatgt aaggaggtgg tgggatgcta tttctgaccg
gagttcttgg aa - #gaaagtgc 660 - - tgaggtatat gaagagcgtg gaggagaaga
acaaacaaga agaactcaag aa - #gcagcagc 720 - - agcagcagga agaggctcct
agaacctcca ccgacccaac tcgggtagac tc - #gagaaagc 780 - - agagcagaac
agagcctcgg acaatattgg ttcctcctgc tgataacgag tc - #atcagctt 840 - -
cgatagttcc tcgaacaaag aagcctcttc ctggtgatca cttagtgtct ac -
#tcaacaaa 900 - - ttgatggtgt tggtatgcca gccacaaatt gatggtgatg
gtcgtcttag tg - #gtgtttgt 960 - - cttgtctttt attgtttggt tctttaacaa
gagttatatt tttaccatca aa - #aaaaaaaa 1020 - - aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 1080 - - aaaaaaaaaa
aaaaaaaa - # - # - #1098 - - - - <210> SEQ ID NO 36
<211> LENGTH: 300 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 36 - - Met Glu Gly Leu Arg Ile Gly
Ala Pro Ile Me - #t Gln Val Tyr His Glu 1 5 - # 10 - # 15 - - Lys
Ser Phe Ile Leu Pro Asp Val Ser Arg Va - #l Leu Ala Cys Leu Tyr 20
- # 25 - # 30 - - Glu Lys Asp Val Lys Phe Glu Thr His Thr Al - #a
Ser Tyr Arg Ser Leu 35 - # 40 - # 45 - - Leu Gly Leu Gln Ala Ser
Ser His Ala Pro Va - #l Pro Phe Tyr Glu Gly 50 - # 55 - # 60 - -
Pro Thr Phe Leu Glu Glu Ser Arg Glu Ile Cy - #s Arg Tyr Ile Ala Glu
65 - # 70 - # 75 - # 80 - - Lys Tyr Glu Asn Gln Gly Tyr Pro Phe Leu
Le - #u Gly Lys Asp Ala Leu 85 - # 90 - # 95 - - Glu Arg Ala Ser
Ile Glu Gln Trp Leu His As - #n Glu Glu His Ala Phe 100 - # 105 - #
110 - - Asn Pro Pro Ser Arg Ala Leu Phe Phe His Le - #u Ala Phe Pro
Leu Gly 115 - # 120 - # 125 - - Glu Gly Glu Asp Asp Asp Ile Asp Val
His Th - #r Arg Lys Leu Glu Glu 130 - # 135 - # 140 - - Val Leu Glu
Val Tyr Glu Gln Arg Leu Ser As - #p Ser Glu Phe Leu Val 145 1 - #50
1 - #55 1 - #60 - - Gly Asn Lys Phe Thr Leu Ala Asp Leu Val Hi - #s
Leu Pro Asn Ser His 165 - # 170 - # 175 - - Tyr Ile Lys Ala Ser Asn
Lys Phe Leu Tyr Le - #u Tyr Asp Ser Arg Lys 180 - # 185 - # 190 - -
Asn Val Arg Arg Trp Trp Asp Ala Ile Ser As - #p Arg Ser Ser Trp Lys
195 - # 200 - # 205 - - Lys Val Leu Arg Tyr Met Lys Ser Val Glu Gl
- #u Lys Asn Lys Gln Glu 210 - # 215 - # 220 - - Glu Leu Lys Lys
Gln Gln Gln Gln Gln Glu Gl - #u Ala Pro Arg Thr Ser 225 2 - #30 2 -
#35 2 - #40 - - Thr Asp Pro Thr Arg Val Asp Ser Arg Lys Gl - #n Ser
Arg Thr Glu Pro 245 - # 250 - # 255 - - Arg Thr Ile Leu Val Pro Pro
Ala Asp Asn Gl - #u Ser Ser Ala Ser Ile 260 - # 265 - # 270 - - Val
Pro Arg Thr Lys Lys Pro Leu Pro Gly As - #p His Leu Val Ser Thr 275
- # 280 - # 285 - - Gln Gln Ile Asp Gly Val Gly Met Pro Ala Th - #r
Asn 290 - # 295 - # 300 - - - - <210> SEQ ID NO 37
<211> LENGTH: 937 <212> TYPE: DNA <213> ORGANISM:
maize - - <400> SEQUENCE: 37 - - gttccgctcc gccacaccaa
aaagaaacaa aagcctacgg cgatcgatcg ag - #aagctggt 60 - - catcgtcagg
atgtcatcgc cgcagtcagc agcgccgccc gtgaagctga tc - #acggcgtt 120 - -
cggcagcccg ttcgcccacc gcgtggaggt ggcgctcgct ctcaaggggg tg -
#ccgtacga 180 - - gctggtcgtg gaggacctag ccaacaagag cgagctgctg
ctcacgcaca ac - #ccagtcca 240 - - ccagtcggtc cctgtcctcc tccacggtga
ccgcgctgtc tgcgagtccc tc - #gtcatcgt 300 - - cgagtacgtc gacgagacct
tccaccatgg cgcggcgccg gggatcctcc cg - #gccgaccc 360 - - ctacgaccgc
gccaccgccc gcttctgggc tgacttcatc gacaacaagt gc - #ttgaagcc 420 - -
gatgtggctg tcgatgtgga cggacggcga ggcgcaggcg cggttcgtca gg -
#gagacgaa 480 - - ggagagcctg ggggtgctgg acgcgcaact ccaggggaag
aggttcttcg cc - #ggcgacgc 540 - - gctcggcttc gtcgacctcg ccgcctgcac
gctggctcac tggctaggcg tg - #ctggagga 600 - - agtggccgga gtgcacctga
tagcggcgga cggcgagtac cccgctctgc gc - #cgctgggc 660 - - caaggagtac
gtctccgatg aggtcgtgag ccggtcgctg ccggacaggg ac - #gagctcgt 720 - -
cgccttcttc accgccagca aggagaggta caagtcgtgg gtcagggcag ag -
#gtggagcg 780 - - acattgatcg ctaaaaatga cgctggttgt tgaactctgt
acgttctctt gt - #agtcgaat 840 - - aatgtgtgga tcgttatgta tacgtactac
gtatttcatc gtgttatata ca - #tctaataa 900 - - gacatgcaag ctcgatcaaa
aaaaaaaaaa aaaaaaa - # - # 937 - - - - <210> SEQ ID NO 38
<211> LENGTH: 238 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 38 - - Met Ser Ser Pro Gln Ser Ala
Ala Pro Pro Va - #l Lys Leu Ile Thr Ala 1 5 - # 10 - # 15 - - Phe
Gly Ser Pro Phe Ala His Arg Val Glu Va - #l Ala Leu Ala Leu Lys 20
- # 25 - # 30 - - Gly Val Pro Tyr Glu Leu Val Val Glu Asp Le - #u
Ala Asn Lys Ser Glu 35 - # 40 - # 45 - - Leu Leu Leu Thr His Asn
Pro Val His Gln Se - #r Val Pro Val Leu Leu 50 - # 55 - # 60 - -
His Gly Asp Arg Ala Val Cys Glu Ser Leu Va - #l Ile Val Glu Tyr Val
65 - # 70 - # 75 - # 80 - - Asp Glu Thr Phe His His Gly Ala Ala Pro
Gl - #y Ile Leu Pro Ala Asp 85 - # 90 - # 95 - - Pro Tyr Asp Arg
Ala Thr Ala Arg Phe Trp Al - #a Asp Phe Ile Asp Asn 100 - # 105 - #
110 - - Lys Cys Leu Lys Pro Met Trp Leu Ser Met Tr - #p Thr Asp Gly
Glu Ala 115 - # 120 - # 125 - - Gln Ala Arg Phe Val Arg Glu Thr Lys
Glu Se - #r Leu Gly Val Leu Asp 130 - # 135 - # 140 - - Ala Gln Leu
Gln Gly Lys Arg Phe Phe Ala Gl - #y Asp Ala Leu Gly Phe 145 1 - #50
1 - #55 1 - #60 - - Val Asp Leu Ala Ala Cys Thr Leu Ala His Tr - #p
Leu Gly Val Leu Glu 165 - # 170 - # 175 - - Glu Val Ala Gly Val His
Leu Ile Ala Ala As - #p Gly Glu Tyr Pro Ala 180 - # 185 - # 190 - -
Leu Arg Arg Trp Ala Lys Glu Tyr Val Ser As - #p Glu Val Val Ser Arg
195 - # 200 - # 205 - - Ser Leu Pro Asp Arg Asp Glu Leu Val Ala Ph
- #e Phe Thr Ala Ser Lys 210 - # 215 - # 220 - - Glu Arg Tyr Lys
Ser Trp Val Arg Ala Glu Va - #l Glu Arg His 225 2 - #30 2 - #35 - -
- - <210> SEQ ID NO 39 <211> LENGTH: 773 <212>
TYPE: DNA <213> ORGANISM: maize - - <400> SEQUENCE: 39
- - cgggagccga cgacctgaag gtgctgggcc tgtggacgag cccgttcgtg at -
#ccgggtcc 60 - - gcatcgtgct caacctcaag ggcctggcgt acgagtacgt
ggaggacgac ct - #cggcaaca 120 - - agagcgcgct cctgctcagc tccaacccgg
tgcacaagac cgtgcccgtg ct - #gctccacg 180 - - cgggtcgccc cgtaaacgag
tcccagatca tcctgcagta catcgacgag gt - #ctgggcgg 240 - - ggaccgggcc
ggccgtgctg ccgcgcgacc cctatgagcg cgcggccgcg cg - #gttctggg 300 - -
cggcctacat cgacgacaag gtgaagtccg cgtggctggg catgctgttc ga -
#gtgcaggg 360 - - acgaggggga gcgggcggag gcggtggcgc gggccggcga
ggcgctcggg ac - #gctggagg 420 - - gcgcgctcag ggggaagccc ttcttcggcg
gcgacggcgt cggcttcgtg ga - #cgccgtgc 480 - - tcggcgggta cctcggctgg
ttcggggccg tcggcaggat catcggccgc ag - #gctgatcg 540 - - acccgactaa
gacgccgctg ctggccgcgt gggaggaccg gttccgcgcc gc - #cgacgtgg 600 - -
ccaagggcgt cgtaccggac gacgtcgaca agatgctcgc gttcctggag ac -
#cctgctcg 660 - - cgaactacta ctccaagtga ctgtactgag agcgaactac
tgctccaagt ga - #ctaaataa 720 - - gaagggctgc ttaattaata attacagtat
ataaaaaaaa aaaaaaaaaa aa - #a 773 - - - - <210> SEQ ID NO 40
<211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 40 - - Gly Ala Asp Asp Leu Lys Val
Leu Gly Leu Tr - #p Thr Ser Pro Phe Val 1 5 - # 10 - # 15 - - Ile
Arg Val Arg Ile Val Leu Asn Leu Lys Gl - #y Leu Ala Tyr Glu Tyr 20
- # 25 - # 30 - - Val Glu Asp Asp Leu Gly Asn Lys Ser Ala Le - #u
Leu Leu Ser Ser Asn 35 - # 40 - # 45
- - Pro Val His Lys Thr Val Pro Val Leu Leu Hi - #s Ala Gly Arg Pro
Val 50 - # 55 - # 60 - - Asn Glu Ser Gln Ile Ile Leu Gln Tyr Ile As
- #p Glu Val Trp Ala Gly 65 - # 70 - # 75 - # 80 - - Thr Gly Pro
Ala Val Leu Pro Arg Asp Pro Ty - #r Glu Arg Ala Ala Ala 85 - # 90 -
# 95 - - Arg Phe Trp Ala Ala Tyr Ile Asp Asp Lys Va - #l Lys Ser
Ala Trp Leu 100 - # 105 - # 110 - - Gly Met Leu Phe Glu Cys Arg Asp
Glu Gly Gl - #u Arg Ala Glu Ala Val 115 - # 120 - # 125 - - Ala Arg
Ala Gly Glu Ala Leu Gly Thr Leu Gl - #u Gly Ala Leu Arg Gly 130 - #
135 - # 140 - - Lys Pro Phe Phe Gly Gly Asp Gly Val Gly Ph - #e Val
Asp Ala Val Leu 145 1 - #50 1 - #55 1 - #60 - - Gly Gly Tyr Leu Gly
Trp Phe Gly Ala Val Gl - #y Arg Ile Ile Gly Arg 165 - # 170 - # 175
- - Arg Leu Ile Asp Pro Thr Lys Thr Pro Leu Le - #u Ala Ala Trp Glu
Asp 180 - # 185 - # 190 - - Arg Phe Arg Ala Ala Asp Val Ala Lys Gly
Va - #l Val Pro Asp Asp Val 195 - # 200 - # 205 - - Asp Lys Met Leu
Ala Phe Leu Glu Thr Leu Le - #u Ala Asn Tyr Tyr Ser 210 - # 215 - #
220 - - Lys 225 - - - - <210> SEQ ID NO 41 <211>
LENGTH: 860 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 41 - - agaaaaaagc ataagctgag catccatcaa
tggcggatgc tggcaacgag gc - #cgagggtc 60 - - tgacgctgtt gggcctgcac
gtgagcccct tcgcgttgcg cgtgcgcatg gc - #gctgagcc 120 - - tcaagggcct
gagctacgag tacatcgagc aggacctgtt ccacaagggc ga - #gctcctcc 180 - -
tcagctcaaa ccccgtgcac aagaaggtgc ccgtgctcat ccaccacggc aa -
#gcccatct 240 - - gcgagtccct cgccgtcgtg gagtacgtcg atgaggtctg
gcccggcgcc gc - #cgccacca 300 - - tcctccccgc cgacccccac ggtcgcgcca
ccgctcgctt ctgggccgcc ta - #catcgacg 360 - - gcaagctgtt tccggcgtgg
acagggatca tgaaggcggc gacggaggaa gc - #gagggcgg 420 - - ataagctgag
ggagacgcac gccgcggtcc tcaacctgga gaaggccttc gc - #cgagatca 480 - -
gctctagctc cagcaacgac ggcgcggcct tcttcggcgg cgactccgtc gg -
#gtacctgg 540 - - acctcgcgct cgggtgctcc ctgccgtggt tcggggcgct
gcgcgccatg ct - #cggcgtcg 600 - - agatcatcga cgccgcccag gctccgctcc
tggtggcgtg ggccgagcga tt - #tggggaga 660 - - ccccggtggc caaggaggtg
ctgccgcagc cggacgaggc tgtggcctac gc - #caagaaga 720 - - ttcaggccta
ctgggcttct gctaagaact gatgagcacc gaatcctgtc at - #gatgaaat 780 - -
tgaagcagca atacttgtat aacactccaa tcatggtgaa taaaggcctc ta -
#aactgttg 840 - - gttaataaaa aaaaaaaaaa - # - # - #860 - - - -
<210> SEQ ID NO 42 <211> LENGTH: 240 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 42 - -
Met Ala Asp Ala Gly Asn Glu Ala Glu Gly Le - #u Thr Leu Leu Gly Leu
1 5 - # 10 - # 15 - - His Val Ser Pro Phe Ala Leu Arg Val Arg Me -
#t Ala Leu Ser Leu Lys 20 - # 25 - # 30 - - Gly Leu Ser Tyr Glu Tyr
Ile Glu Gln Asp Le - #u Phe His Lys Gly Glu 35 - # 40 - # 45 - -
Leu Leu Leu Ser Ser Asn Pro Val His Lys Ly - #s Val Pro Val Leu Ile
50 - # 55 - # 60 - - His His Gly Lys Pro Ile Cys Glu Ser Leu Al -
#a Val Val Glu Tyr Val 65 - # 70 - # 75 - # 80 - - Asp Glu Val Trp
Pro Gly Ala Ala Ala Thr Il - #e Leu Pro Ala Asp Pro 85 - # 90 - #
95 - - His Gly Arg Ala Thr Ala Arg Phe Trp Ala Al - #a Tyr Ile Asp
Gly Lys 100 - # 105 - # 110 - - Leu Phe Pro Ala Trp Thr Gly Ile Met
Lys Al - #a Ala Thr Glu Glu Ala 115 - # 120 - # 125 - - Arg Ala Asp
Lys Leu Arg Glu Thr His Ala Al - #a Val Leu Asn Leu Glu 130 - # 135
- # 140 - - Lys Ala Phe Ala Glu Ile Ser Ser Ser Ser Se - #r Asn Asp
Gly Ala Ala 145 1 - #50 1 - #55 1 - #60 - - Phe Phe Gly Gly Asp Ser
Val Gly Tyr Leu As - #p Leu Ala Leu Gly Cys 165 - # 170 - # 175 - -
Ser Leu Pro Trp Phe Gly Ala Leu Arg Ala Me - #t Leu Gly Val Glu Ile
180 - # 185 - # 190 - - Ile Asp Ala Ala Gln Ala Pro Leu Leu Val Al
- #a Trp Ala Glu Arg Phe 195 - # 200 - # 205 - - Gly Glu Thr Pro
Val Ala Lys Glu Val Leu Pr - #o Gln Pro Asp Glu Ala 210 - # 215 - #
220 - - Val Ala Tyr Ala Lys Lys Ile Gln Ala Tyr Tr - #p Ala Ser Ala
Lys Asn 225 2 - #30 2 - #35 2 - #40 - - - - <210> SEQ ID NO
43 <211> LENGTH: 1228 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 43 - - gtcgtcactg
ctgaccatag gtggccggcc ggccgaacaa accctcggca cg - #atcgcctg 60 - -
cctccataaa tctccctctt cacttcaggc gaaaaggatc aaccaaaccc tc -
#taatccat 120 - - ttcggcattt ccaacgcctt cgccctacca gccacgtcgc
ttcgaggccg at - #cgaccgag 180 - - cagctggtgg caatggcggc ggcggcggag
gtcgtgctgc tggacttctg gg - #tgagcccc 240 - - ttcgggcagc gctgccggat
cgcgctggcg gagaagggcg tggcctacga gt - #accgcgag 300 - - caggacctcc
tggacaaggg cgagctgctc ctccgctcca accccatcca ca - #agaagatc 360 - -
cccgtcctgc tccacgccgg caggcccgtc tgcgagtcgc tcgtcatcct cc -
#agtacatc 420 - - gacgaggcct ggccggacgt cgcgccgctc ctccccaagg
acgaccccta cg - #cccgcgcg 480 - - caggcgcgtt tctgggccga ttacatcgac
aagaagatct atgacagcca ga - #ctcggctg 540 - - tggaagttcg agggcgaggc
gcgggagcag gcgaagaagg acctggtgga gg - #tcctggag 600 - - acctggaggg
ggagctcgcc gacaagcctt tcttcggcgg cggcgccctc gg - #cttcgtgg 660 - -
acgtggctct ggtgcccttc acgtcctggt tcctcgccta cgagaagctg gg -
#cgggttca 720 - - gcgtccagga gcactgcccc aggatcgtgg cctgggccgc
gcgctgcagg ga - #gcgggaga 780 - - gcgtggccaa ggccatgtcc gaccctgcca
aggtgctcga gttcgtccag tt - #cctccaga 840 - - gcaagttcgg ggccaagtga
tcggaagcat tgcgtgtgct gctagcctgc ta - #tgccctat 900 - - gcaggccagg
ctggtgcttt gatctgctcg atcagctcta tgcccatgct ag - #cgttgcat 960 - -
agcgcagttg atgtgtgatg tgtctggttg gttgtagctg ctctttgcct gg -
#tttcgtac 1020 - - gtcagtgtaa ggtttcaggt tttcagtgtc tggggtagct
ctgcgttgcc ct - #tgcccctg 1080 - - ccccctacct agcggctctt gagctcttcg
gctcgccagc aataaagttg ca - #gaggcttt 1140 - - agctaaaagt ttctgtattt
tttagttgac gattattggt ccaatgtatt cg - #ggaatttt 1200 - - gttctctcta
aaaaaaaaaa aaaaaaaa - # - # 1228 - - - - <210> SEQ ID NO 44
<211> LENGTH: 230 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 44 - - Met Ala Ala Ala Ala Glu Val
Val Leu Leu As - #p Phe Trp Val Ser Pro 1 5 - # 10 - # 15 - - Phe
Gly Gln Arg Cys Arg Ile Ala Leu Ala Gl - #u Lys Gly Val Ala Tyr 20
- # 25 - # 30 - - Glu Tyr Arg Glu Gln Asp Leu Leu Asp Lys Gl - #y
Glu Leu Leu Leu Arg 35 - # 40 - # 45 - - Ser Asn Pro Ile His Lys
Lys Ile Pro Val Le - #u Leu His Ala Gly Arg 50 - # 55 - # 60 - -
Pro Val Cys Glu Ser Leu Val Ile Leu Gln Ty - #r Ile Asp Glu Ala Trp
65 - # 70 - # 75 - # 80 - - Pro Asp Val Ala Pro Leu Leu Pro Lys Asp
As - #p Pro Tyr Ala Arg Ala 85 - # 90 - # 95 - - Gln Ala Arg Phe
Trp Ala Asp Tyr Ile Asp Ly - #s Lys Ile Tyr Asp Ser 100 - # 105 - #
110 - - Gln Thr Arg Leu Trp Lys Phe Glu Gly Glu Al - #a Arg Glu Gln
Ala Lys 115 - # 120 - # 125 - - Lys Asp Leu Val Glu Val Leu Glu Thr
Trp Ar - #g Gly Ser Ser Pro Thr 130 - # 135 - # 140 - - Ser Leu Ser
Ser Ala Ala Ala Pro Ser Ala Se - #r Trp Thr Trp Leu Trp 145 1 - #50
1 - #55 1 - #60 - - Cys Pro Ser Arg Pro Gly Ser Ser Pro Thr Ar - #g
Ser Trp Ala Gly Ser 165 - # 170 - # 175 - - Ala Ser Arg Ser Thr Ala
Pro Gly Ser Trp Pr - #o Gly Pro Arg Ala Ala 180 - # 185 - # 190 - -
Gly Ser Gly Arg Ala Trp Pro Arg Pro Cys Pr - #o Thr Leu Pro Arg Cys
195 - # 200 - # 205 - - Ser Ser Ser Ser Ser Ser Ser Arg Ala Ser Se
- #r Gly Pro Ser Asp Arg 210 - # 215 - # 220 - - Lys His Cys Val
Cys Cys 225 2 - #30 - - - - <210> SEQ ID NO 45 <211>
LENGTH: 840 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 45 - - caagctaagc aagtgccaac caacgagtag
caggaaacat gtctccgccc gt - #caagatcc 60 - - tcggccacta cgcgagcccg
tactcgcacc gcgtcgaggc cgctctgcgg ct - #caagggcg 120 - - tgccgtacga
gctggtccag gaagacctgg gcaacaagag cgagctgctg ct - #cgccaaga 180 - -
accctgtcca caagaaggtg cccgtgctcc tccatggcga cagggccgtc tg -
#cgagtccc 240 - - tcctcatcgt cgagtacgtc gacgaggcct tcgacgggcc
gtccatcctg cc - #ggccgacc 300 - - cccacgaccg tgccgtcgcc cgtttctggg
cgaacttctt ggacaccaag tt - #ctcccagc 360 - - cgttctggct ggcgtactgg
gcggagggcg aggcgcagaa ggccgtggtg aa - #ggaggcca 420 - - aggagaacct
ggcgctcctg gaggcgcagc tcggcgggaa gaggttcttc gg - #cggcgaca 480 - -
cgcccgggta cctcgacata gccgcgtgca cgttgggtcc ttggatcggc gt -
#gctcgagg 540 - - aggtgactgg agtggccttg ctggacgccg acgagttccc
cgctctatgc ca - #gtgggcca 600 - - gggactacag ctccagtgaa gcgctcaggc
catgcctgcc ggacagggac cg - #actcgttg 660 - - cctacttcac cgagaacaag
gagaagtaca agacatttgc caaggcaacg tt - #gcatcagt 720 - - agctgctagt
tgggtgcaaa ccgcttgttt atctctgtgt ggaataatgt at - #acgtacgt 780 - -
gctccctcga tatcaaataa atcagctacc ggagttgact gtagtcaaaa aa -
#aaaaaaaa 840 - - - - <210> SEQ ID NO 46 <211> LENGTH:
227 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 46 - - Met Ser Pro Pro Val Lys Ile Leu Gly
His Ty - #r Ala Ser Pro Tyr Ser 1 5 - # 10 - # 15 - - His Arg Val
Glu Ala Ala Leu Arg Leu Lys Gl - #y Val Pro Tyr Glu Leu 20 - # 25 -
# 30 - - Val Gln Glu Asp Leu Gly Asn Lys Ser Glu Le - #u Leu Leu
Ala Lys Asn 35 - # 40 - # 45 - - Pro Val His Lys Lys Val Pro Val
Leu Leu Hi - #s Gly Asp Arg Ala Val 50 - # 55 - # 60 - - Cys Glu
Ser Leu Leu Ile Val Glu Tyr Val As - #p Glu Ala Phe Asp Gly 65 - #
70 - # 75 - # 80 - - Pro Ser Ile Leu Pro Ala Asp Pro His Asp Ar -
#g Ala Val Ala Arg Phe 85 - # 90 - # 95 - - Trp Ala Asn Phe Leu Asp
Thr Lys Phe Ser Gl - #n Pro Phe Trp Leu Ala 100 - # 105 - # 110 - -
Tyr Trp Ala Glu Gly Glu Ala Gln Lys Ala Va - #l Val Lys Glu Ala Lys
115 - # 120 - # 125 - - Glu Asn Leu Ala Leu Leu Glu Ala Gln Leu Gl
- #y Gly Lys Arg Phe Phe 130 - # 135 - # 140 - - Gly Gly Asp Thr
Pro Gly Tyr Leu Asp Ile Al - #a Ala Cys Thr Leu Gly 145 1 - #50 1 -
#55 1 - #60 - - Pro Trp Ile Gly Val Leu Glu Glu Val Thr Gl - #y Val
Ala Leu Leu Asp 165 - # 170 - # 175 - - Ala Asp Glu Phe Pro Ala Leu
Cys Gln Trp Al - #a Arg Asp Tyr Ser Ser 180 - # 185 - # 190 - - Ser
Glu Ala Leu Arg Pro Cys Leu Pro Asp Ar - #g Asp Arg Leu Val Ala 195
- # 200 - # 205 - - Tyr Phe Thr Glu Asn Lys Glu Lys Tyr Lys Th - #r
Phe Ala Lys Ala Thr
210 - # 215 - # 220 - - Leu His Gln 225 - - - - <210> SEQ ID
NO 47 <211> LENGTH: 970 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 47 - - acgcaaacgc
aagctcccaa gcagctacca accagccggg agagggagcg ca - #cgcactgc 60 - -
gagaatggcc acggccggcg gaggggccgg gcatctgaag ctgctgggcg cg -
#tggccgag 120 - - ccccttcgtg aaccgtgtac gcatggcgct gcacctcaag
gggctggagt ac - #gagaacgt 180 - - ggaggaggac ctcaccaaca agagcgacct
gctcctcgcc tccaaccccg tc - #cacaagct 240 - - cgttcccgtc ctcctccacg
gcgacaagcc catctccgag tcactcgtca tc - #gtggagta 300 - - cctcgacgac
gccttccccg gcgctggcca ggccgtcctc cccgccgacc cc - #tatgaacg 360 - -
cgccgtcgct cgcttctggg ccaaatacgt cgacggcaag ttgcacggca tg -
#atggtgaa 420 - - ggcgctcatg ggggcaacgg aggaggagag ggcgacggcg
acggtggacg cg - #ctggccgc 480 - - tatggacacg ctggagggcg cgttcgccga
gtgctccggc gggaaaaagt tc - #ttcgccgg 540 - - cgacgcgccc gggtacctgg
acgtcgcgct gggaggcttc atcggctggc tg - #cgcgcctg 600 - - ggacaaggtg
gggggcgtca agctgctgga cgccggccgg gtcccgcgcc tg - #gccacgtg 660 - -
ggcggagcgc ttcgccgcgc tcgacgtagc caaggaggtc atcccggacc cc -
#gaccacat 720 - - cgccgagttt gccaaggtgc tgcaggcacg ctccgcggca
gctgccacca gc - #aactgagc 780 - - tcgcaagtga atgctgctgc tctgccttcc
tgataaaatc atatcatggt ac - #ccggtttg 840 - - ttcgtttaat ctggcgcgga
aaaaacatgg tttgttgtcg taccaaacta ag - #agtatgca 900 - - tgcatgcttg
ttcttaatta tatgtacatt cgttcaagaa aaaaaaaaaa aa - #aaaaaaaa 960 - -
aaaaaaaaaa - # - # - # 970 - - - - <210> SEQ ID NO 48
<211> LENGTH: 237 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 48 - - Met Ala Thr Ala Gly Gly Gly
Ala Gly His Le - #u Lys Leu Leu Gly Ala 1 5 - # 10 - # 15 - - Trp
Pro Ser Pro Phe Val Asn Arg Val Arg Me - #t Ala Leu His Leu Lys 20
- # 25 - # 30 - - Gly Leu Glu Tyr Glu Asn Val Glu Glu Asp Le - #u
Thr Asn Lys Ser Asp 35 - # 40 - # 45 - - Leu Leu Leu Ala Ser Asn
Pro Val His Lys Le - #u Val Pro Val Leu Leu 50 - # 55 - # 60 - -
His Gly Asp Lys Pro Ile Ser Glu Ser Leu Va - #l Ile Val Glu Tyr Leu
65 - # 70 - # 75 - # 80 - - Asp Asp Ala Phe Pro Gly Ala Gly Gln Ala
Va - #l Leu Pro Ala Asp Pro 85 - # 90 - # 95 - - Tyr Glu Arg Ala
Val Ala Arg Phe Trp Ala Ly - #s Tyr Val Asp Gly Lys 100 - # 105 - #
110 - - Leu His Gly Met Met Val Lys Ala Leu Met Gl - #y Ala Thr Glu
Glu Glu 115 - # 120 - # 125 - - Arg Ala Thr Ala Thr Val Asp Ala Leu
Ala Al - #a Met Asp Thr Leu Glu 130 - # 135 - # 140 - - Gly Ala Phe
Ala Glu Cys Ser Gly Gly Lys Ly - #s Phe Phe Ala Gly Asp 145 1 - #50
1 - #55 1 - #60 - - Ala Pro Gly Tyr Leu Asp Val Ala Leu Gly Gl - #y
Phe Ile Gly Trp Leu 165 - # 170 - # 175 - - Arg Ala Trp Asp Lys Val
Gly Gly Val Lys Le - #u Leu Asp Ala Gly Arg 180 - # 185 - # 190 - -
Val Pro Arg Leu Ala Thr Trp Ala Glu Arg Ph - #e Ala Ala Leu Asp Val
195 - # 200 - # 205 - - Ala Lys Glu Val Ile Pro Asp Pro Asp His Il
- #e Ala Glu Phe Ala Lys 210 - # 215 - # 220 - - Val Leu Gln Ala
Arg Ser Ala Ala Ala Ala Th - #r Ser Asn 225 2 - #30 2 - #35 - - - -
<210> SEQ ID NO 49 <211> LENGTH: 756 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 49 - -
cggcggtgag gctggtgggc tccttcgcca gcccgttcgt ccaccgcgcc ga -
#ggtggccc 60 - - tgcgcctcaa aggggtgccc tacgagctca tcctggagga
cctgggcaac aa - #gagcgagc 120 - - tgctgctggc acacaacccc gtgcacaaac
tcgtgcccgt gctcctccac gg - #cgacaggg 180 - - ccatctccga gtcgctcgtc
atcctcgagt acgtcgacga ggccttcgac gg - #gccgcctc 240 - - tcctccccgc
ggaaccccac gcgagggcgg acgcgcggtt ctgggcccac tt - #catcgacc 300 - -
aaaagttcgc gcggccgttc tggatgtcgt tctggacgga cgacgaggag cg -
#cagggagg 360 - - ctatggcgaa ggaggccaag gagaacctgg ctctgctcga
ggcgcagctc ag - #ggggcaga 420 - - ggttcttcgg cggcgaggcc atcggcttcg
tcgacatcgc cgcctgtgcg ct - #ggcgcact 480 - - gggtcggggt catcgaggag
gctgccgggg tggtcctcgt cggcggcgag ga - #gttcccag 540 - - cgctccgcga
gtgggccgac gcctacgtca acgacgccac cgtgaagcag tg - #cttgagga 600 - -
gccgcgacga gctcgtcgat tacttctccg ccaggaagga gatgtacttg ct -
#gcgagcga 660 - - gggccactcc gcgcagctga tctggacccc atgtttcctt
ccgttcgcaa ta - #agccaata 720 - - ataaagacta gtttggtaaa aaaaaaaaaa
aaaaaa - # - # 756 - - - - <210> SEQ ID NO 50 <211>
LENGTH: 225 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 50 - - Ala Val Arg Leu Val Gly Ser Phe Ala
Ser Pr - #o Phe Val His Arg Ala 1 5 - # 10 - # 15 - - Glu Val Ala
Leu Arg Leu Lys Gly Val Pro Ty - #r Glu Leu Ile Leu Glu 20 - # 25 -
# 30 - - Asp Leu Gly Asn Lys Ser Glu Leu Leu Leu Al - #a His Asn
Pro Val His 35 - # 40 - # 45 - - Lys Leu Val Pro Val Leu Leu His
Gly Asp Ar - #g Ala Ile Ser Glu Ser 50 - # 55 - # 60 - - Leu Val
Ile Leu Glu Tyr Val Asp Glu Ala Ph - #e Asp Gly Pro Pro Leu 65 - #
70 - # 75 - # 80 - - Leu Pro Ala Glu Pro His Ala Arg Ala Asp Al -
#a Arg Phe Trp Ala His 85 - # 90 - # 95 - - Phe Ile Asp Gln Lys Phe
Ala Arg Pro Phe Tr - #p Met Ser Phe Trp Thr 100 - # 105 - # 110 - -
Asp Asp Glu Glu Arg Arg Glu Ala Met Ala Ly - #s Glu Ala Lys Glu Asn
115 - # 120 - # 125 - - Leu Ala Leu Leu Glu Ala Gln Leu Arg Gly Gl
- #n Arg Phe Phe Gly Gly 130 - # 135 - # 140 - - Glu Ala Ile Gly
Phe Val Asp Ile Ala Ala Cy - #s Ala Leu Ala His Trp 145 1 - #50 1 -
#55 1 - #60 - - Val Gly Val Ile Glu Glu Ala Ala Gly Val Va - #l Leu
Val Gly Gly Glu 165 - # 170 - # 175 - - Glu Phe Pro Ala Leu Arg Glu
Trp Ala Asp Al - #a Tyr Val Asn Asp Ala 180 - # 185 - # 190 - - Thr
Val Lys Gln Cys Leu Arg Ser Arg Asp Gl - #u Leu Val Asp Tyr Phe 195
- # 200 - # 205 - - Ser Ala Arg Lys Glu Met Tyr Leu Leu Arg Al - #a
Arg Ala Thr Pro Arg 210 - # 215 - # 220 - - Ser 225 - - - -
<210> SEQ ID NO 51 <211> LENGTH: 967 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 51 - -
cagccacggc gagcaagcga tgatgagcgg cggcgcggtg aaggtgatcg gc -
#gccctgga 60 - - cagcccgttc agccaccgcg cggaggcggc gctgcgcctc
aagggagtcc cc - #tacgagct 120 - - tgtcctggag aaggacctgc gcgacaaaag
cgagctgctg ctgcggcaca ac - #cccgtcca 180 - - caagaaggtg cccgtgctcc
ttcacggcgg ccgccgcgcc gtctgcgagt cg - #ctcgtcat 240 - - cgtcgagtac
gttgacgagg cattccgcgg cccgccactc ctccccgccg ac - #ccctccgc 300 - -
ccgcgccgcc gcccgcttct gggcccgctt catcgacgac aagtgctcga cg -
#cccttctg 360 - - gctggcgatg tggacggagg gcgaggcgca gagggggttc
gtgaaggaga tc - #aaggagaa 420 - - cctgaagctg ctggaggggc aggtgaaggg
caagcggttc ttcggcggcg gc - #gacgtggg 480 - - ctacctcgac gtcgccgcca
gcgtgttcgc gcactggctt ccggtctgcg ag - #gaggtcgc 540 - - gggcgtcagc
ctggtcacgg ccgaggagta cccggacctg tgccggtggg cg - #agggagta 600 - -
cacctcccac gacgccgtca agcggtgcct gcctggcagg gaggagctgc tc -
#gcccgttt 660 - - cagcgccagg aaggactcgt ttgtggccgc ggcgaggtca
atggcgccgg cg - #ccggagaa 720 - - gtaatctatg ggaattcaac tgggtgcatg
gatagcataa atttaagtat tg - #tgacaagt 780 - - ggtcaggact gctgtcacgt
accgtcgagc ccgggagata tgtactctcc gg - #ctcaggca 840 - - aatgccatct
gtcctgatgc atgtgttttg ttcctacttt gtttgactgc tt - #gttgaata 900 - -
aaaaaagata tccggttgtg ttcaaaaaaa aaaaaaaaaa aaaaaaaaaa aa -
#aaaaaaaa 960 - - aaaaaaa - # - # - # 967 - - - - <210> SEQ
ID NO 52 <211> LENGTH: 234 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 52 - - Met Met Ser Gly
Gly Ala Val Lys Val Ile Gl - #y Ala Leu Asp Ser Pro 1 5 - # 10 - #
15 - - Phe Ser His Arg Ala Glu Ala Ala Leu Arg Le - #u Lys Gly Val
Pro Tyr 20 - # 25 - # 30 - - Glu Leu Val Leu Glu Lys Asp Leu Arg
Asp Ly - #s Ser Glu Leu Leu Leu 35 - # 40 - # 45 - - Arg His Asn
Pro Val His Lys Lys Val Pro Va - #l Leu Leu His Gly Gly 50 - # 55 -
# 60 - - Arg Arg Ala Val Cys Glu Ser Leu Val Ile Va - #l Glu Tyr
Val Asp Glu 65 - # 70 - # 75 - # 80 - - Ala Phe Arg Gly Pro Pro Leu
Leu Pro Ala As - #p Pro Ser Ala Arg Ala 85 - # 90 - # 95 - - Ala
Ala Arg Phe Trp Ala Arg Phe Ile Asp As - #p Lys Cys Ser Thr Pro 100
- # 105 - # 110 - - Phe Trp Leu Ala Met Trp Thr Glu Gly Glu Al - #a
Gln Arg Gly Phe Val 115 - # 120 - # 125 - - Lys Glu Ile Lys Glu Asn
Leu Lys Leu Leu Gl - #u Gly Gln Val Lys Gly 130 - # 135 - # 140 - -
Lys Arg Phe Phe Gly Gly Gly Asp Val Gly Ty - #r Leu Asp Val Ala Ala
145 1 - #50 1 - #55 1 - #60 - - Ser Val Phe Ala His Trp Leu Pro Val
Cys Gl - #u Glu Val Ala Gly Val 165 - # 170 - # 175 - - Ser Leu Val
Thr Ala Glu Glu Tyr Pro Asp Le - #u Cys Arg Trp Ala Arg 180 - # 185
- # 190 - - Glu Tyr Thr Ser His Asp Ala Val Lys Arg Cy - #s Leu Pro
Gly Arg Glu 195 - # 200 - # 205 - - Glu Leu Leu Ala Arg Phe Ser Ala
Arg Lys As - #p Ser Phe Val Ala Ala 210 - # 215 - # 220 - - Ala Arg
Ser Met Ala Pro Ala Pro Glu Lys 225 2 - #30 - - - - <210> SEQ
ID NO 53 <211> LENGTH: 1100 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 53 - - acctcagaca
ctctgcatat atcctctgcg cgtgtatctc gtcgaacaga gc - #cgaaagct 60 - -
ggagcttcca atggcgggag gcaacgacct gaaggtgctc ggcgtgtgga cg -
#agcccgtt 120 - - cgtgatccgg gtccgcatcg tgctcaacct gaagggcctg
gcgtacgagt ac - #gtggagga 180 - - ggacctcggc aacaagagcg cgctcctcct
gggatccaac ccggtgcaca ag - #agcgtgcc 240 - - ggtgctcctc cacgccggcc
gcgccataaa cgagtcccag gtcatcctgc ag - #tacatcga 300 - - cgaggtgtgg
gcggggacgg ggccggccgt gcttccggcc gacccctacg ag - #cgcgcggt 360 - -
ggcgcggttc tggggcgcgt acatcgacga caaggtggag tcggcgtggc tg -
#gggatgct 420 - - gttcaggtgc gcgaacgagg aggagagggc ggcggcggtg
gcgcgcgccc gc - #gaggcgct 480 - - cgacgcgctg gagggcgcgt tccgggactg
ctccaggggg aggccgttct tc - #ggcggcga 540 - - cgacatcggg ttcgtggacg
ccgttctcgg cgggtacctc ggctggttcg gg - #gccgtcgg 600 - - caggatcatc
gggagcaggc tcatcgaccc ggcccggacg ccgctgctgg cc -
#gcgtggga 660 - - ggaccggttc cgcgccgccg acgtggccaa gggcgtcgtg
cccgacgacc tc - #gacaagat 720 - - gctcgcgttc ctgcagaccc tgcgcgctat
gaactacgcc aagtgagagt gt - #ttcgtcgc 780 - - atgaacgtgt gccgtgccgt
gcacgaccta tgatcagttc atgtcgatac gt - #ctatcact 840 - - cagttttgct
tcttccgtca ataatcggtg tgctgaatac atgtacaaca gc - #tgcctata 900 - -
attttgcttc tttcttctaa tacctccgtt tttaatttga tagttcaact tt -
#ataataac 960 - - aaatgtcaaa tttaaaaaaa aaatcggaag gagtattttt
ttaatacatc ca - #agtccatc 1020 - - caataaaagt gctcgtgggg ctttctatta
aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 1080 - - aaaaaaaaaa aaaaaaaaaa
- # - # 110 - #0 - - - - <210> SEQ ID NO 54 <211>
LENGTH: 231 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 54 - - Met Ala Gly Gly Asn Asp Leu Lys Val
Leu Gl - #y Val Trp Thr Ser Pro 1 5 - # 10 - # 15 - - Phe Val Ile
Arg Val Arg Ile Val Leu Asn Le - #u Lys Gly Leu Ala Tyr 20 - # 25 -
# 30 - - Glu Tyr Val Glu Glu Asp Leu Gly Asn Lys Se - #r Ala Leu
Leu Leu Gly 35 - # 40 - # 45 - - Ser Asn Pro Val His Lys Ser Val
Pro Val Le - #u Leu His Ala Gly Arg 50 - # 55 - # 60 - - Ala Ile
Asn Glu Ser Gln Val Ile Leu Gln Ty - #r Ile Asp Glu Val Trp 65 - #
70 - # 75 - # 80 - - - - Ala Gly Thr Gly Pro Ala Val Leu Pro Ala As
- #p Pro Tyr Glu Arg Ala 85 - # 90 - # 95 - - Val Ala Arg Phe Trp
Gly Ala Tyr Ile Asp As - #p Lys Val Glu Ser Ala 100 - # 105 - # 110
- - Trp Leu Gly Met Leu Phe Arg Cys Ala Asn Gl - #u Glu Glu Arg Ala
Ala 115 - # 120 - # 125 - - Ala Val Ala Arg Ala Arg Glu Ala Leu Asp
Al - #a Leu Glu Gly Ala Phe 130 - # 135 - # 140 - - Arg Asp Cys Ser
Arg Gly Arg Pro Phe Phe Gl - #y Gly Asp Asp Ile Gly 145 1 - #50 1 -
#55 1 - #60 - - Phe Val Asp Ala Val Leu Gly Gly Tyr Leu Gl - #y Trp
Phe Gly Ala Val 165 - # 170 - # 175 - - Gly Arg Ile Ile Gly Ser Arg
Leu Ile Asp Pr - #o Ala Arg Thr Pro Leu 180 - # 185 - # 190 - - Leu
Ala Ala Trp Glu Asp Arg Phe Arg Ala Al - #a Asp Val Ala Lys Gly 195
- # 200 - # 205 - - Val Val Pro Asp Asp Leu Asp Lys Met Leu Al - #a
Phe Leu Gln Thr Leu 210 - # 215 - # 220 - - Arg Ala Met Asn Tyr Ala
Lys 225 2 - #30 - - - - <210> SEQ ID NO 55 <211>
LENGTH: 934 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 55 - - acgacggaaa cagtagtgct gccagtagag
agctctcaga actcgggaaa aa - #aatgtcag 60 - - aggccgccgt gcgtgtgatc
ggcctatggc cgagcccgtt cgtgatccgc gt - #cctgatcg 120 - - ccctgaagct
gaagggcgtc gagttcgagt tcgtggagga ggtggtgggc ag - #gaagagcg 180 - -
agctgctgct gaggtcgaac ccggtgcaca agaagatccc cgtcctgctc ca -
#ccacggca 240 - - agcccatctc cgagtctctg atcgtcgtcc agtacatcga
cgaggtctgg tc - #ctccggcg 300 - - cgccggcctt cctccccgtc gacgctcacg
cccgcgccgt ccagcggttc tg - #ggcgcagt 360 - - acgtcgacga caagctgcct
tgggcgatcc gcatactgaa gggaacggac ga - #cgggggca 420 - - tggagcaggc
ggcggggcag ctgtccgcgg ccctgcagct cctagaggag gc - #tttcgcgc 480 - -
agctcagcca ggggaagcgc tacttcggcg gggacagcgt cgggtacctg ga -
#catcgctc 540 - - tggtgtcgca tgtcggctgg gtgaaggcgg tggagaagat
cgccggggtc ac - #cctgctgg 600 - - acaaggccaa ggtcccgaac ctggtggcgt
gggctgatcg tctgtgtgcc ca - #cccggccg 660 - - tggtcgacgc catccctgac
gcggacaagt tcgttgagtt cagcgtcacc ta - #tggctcct 720 - - tttcgaagcc
tatcaatgct cccgccaagt gagcaaaaag ggtccgtgca tg - #ctttcgtc 780 - -
attttcactt tcactgcgcg tgtgccggtg cgtgtcaaaa ttgcatagca ag -
#gggattct 840 - - tctcccacat agatcttctt gtgatcatag tcaccaaatc
agctctgaaa at - #gaagattt 900 - - tctgcccttc caaaggaaaa aaaaaaaaaa
aaaa - # - # 934 - - - - <210> SEQ ID NO 56 <211>
LENGTH: 232 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 56 - - Met Ser Glu Ala Ala Val Arg Val Ile
Gly Le - #u Trp Pro Ser Pro Phe 1 5 - # 10 - # 15 - - Val Ile Arg
Val Leu Ile Ala Leu Lys Leu Ly - #s Gly Val Glu Phe Glu 20 - # 25 -
# 30 - - Phe Val Glu Glu Val Val Gly Arg Lys Ser Gl - #u Leu Leu
Leu Arg Ser 35 - # 40 - # 45 - - Asn Pro Val His Lys Lys Ile Pro
Val Leu Le - #u His His Gly Lys Pro 50 - # 55 - # 60 - - Ile Ser
Glu Ser Leu Ile Val Val Gln Tyr Il - #e Asp Glu Val Trp Ser 65 - #
70 - # 75 - # 80 - - Ser Gly Ala Pro Ala Phe Leu Pro Val Asp Al -
#a His Ala Arg Ala Val 85 - # 90 - # 95 - - Gln Arg Phe Trp Ala Gln
Tyr Val Asp Asp Ly - #s Leu Pro Trp Ala Ile 100 - # 105 - # 110 - -
Arg Ile Leu Lys Gly Thr Asp Asp Gly Gly Me - #t Glu Gln Ala Ala Gly
115 - # 120 - # 125 - - Gln Leu Ser Ala Ala Leu Gln Leu Leu Glu Gl
- #u Ala Phe Ala Gln Leu 130 - # 135 - # 140 - - Ser Gln Gly Lys
Arg Tyr Phe Gly Gly Asp Se - #r Val Gly Tyr Leu Asp 145 1 - #50 1 -
#55 1 - #60 - - Ile Ala Leu Val Ser His Val Gly Trp Val Ly - #s Ala
Val Glu Lys Ile 165 - # 170 - # 175 - - Ala Gly Val Thr Leu Leu Asp
Lys Ala Lys Va - #l Pro Asn Leu Val Ala 180 - # 185 - # 190 - - Trp
Ala Asp Arg Leu Cys Ala His Pro Ala Va - #l Val Asp Ala Ile Pro 195
- # 200 - # 205 - - Asp Ala Asp Lys Phe Val Glu Phe Ser Val Th - #r
Tyr Gly Ser Phe Ser 210 - # 215 - # 220 - - Lys Pro Ile Asn Ala Pro
Ala Lys 225 2 - #30 - - - - <210> SEQ ID NO 57 <211>
LENGTH: 960 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 57 - - aaccgcagct gaagctgctg gccatgtggg
cgagcccgtt tgccctacgg gc - #gaagctag 60 - - cgctcaactt caagggcctg
gcctacgagt acgtagagga ggacctccgc ag - #caagagcg 120 - - acctcctgct
gagctcgaac ccggtgcaca agaaggtgcc cgtcctcatc ca - #caacggcg 180 - -
tgcccgtctg tgagtcgcgg gtcatcgtgg agtacctcga cgaagtctac ag -
#cgccacgg 240 - - gcccccgctt cctccctgcc gacccatacg agcgtgccat
ggcgcgcttc tg - #ggcctcat 300 - - tcatcgacga aaagttcttg gcgtcgtggc
taaaggcagg aaggggcaag ac - #ggacgagg 360 - - agaaggccga agggttgaag
ctgacactcg cggccgtaga aaccttggaa gg - #ggcgttca 420 - - tggagtgctc
caaggggaag cccttctttg gaggcgatag tgtcggctac ct - #ggacatcg 480 - -
cgctcggggc cctggtagcg tggatgcgcg ccaccgaggc gcgtcatggt ct -
#caggctct 540 - - tcgacgcctc cagagtccgc tgctggagaa gtgggtggag
cgcttcagcg ag - #ctggacga 600 - - ggtcgtggcg gtcatgccgg acatcgaccg
gctagtagag ctcggcaagg tg - #agggaggc 660 - - tgctgcggct gcagcagctg
ccgtaaacag ctgaacggaa cgcatctcgc gg - #tattgagg 720 - - cggtcaataa
gtcggagaaa gatcttgata cctgtgtgta acaagcgaat gg - #tgtaataa 780 - -
agaatacaat tagggtgtcg tttagttcac atgtcggtaa cgtaatggat aa -
#ccgataac 840 - - attaaatcat gtttgttata agtccaatcg taatcgatct
catactacaa aa - #aaaaaaaa 900 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 960 - - - - <210> SEQ ID
NO 58 <211> LENGTH: 203 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 58 - - Met Trp Ala Ser
Pro Phe Ala Leu Arg Ala Ly - #s Leu Ala Leu Asn Phe 1 5 - # 10 - #
15 - - Lys Gly Leu Ala Tyr Glu Tyr Val Glu Glu As - #p Leu Arg Ser
Lys Ser 20 - # 25 - # 30 - - Asp Leu Leu Leu Ser Ser Asn Pro Val
His Ly - #s Lys Val Pro Val Leu 35 - # 40 - # 45 - - Ile His Asn
Gly Val Pro Val Cys Glu Ser Ar - #g Val Ile Val Glu Tyr 50 - # 55 -
# 60 - - Leu Asp Glu Val Tyr Ser Ala Thr Gly Pro Ar - #g Phe Leu
Pro Ala Asp 65 - # 70 - # 75 - # 80 - - Pro Tyr Glu Arg Ala Met Ala
Arg Phe Trp Al - #a Ser Phe Ile Asp Glu 85 - # 90 - # 95 - - Lys
Phe Leu Ala Ser Trp Leu Lys Ala Gly Ar - #g Gly Lys Thr Asp Glu 100
- # 105 - # 110 - - Glu Lys Ala Glu Gly Leu Lys Leu Thr Leu Al - #a
Ala Val Glu Thr Leu 115 - # 120 - # 125 - - Glu Gly Ala Phe Met Glu
Cys Ser Lys Gly Ly - #s Pro Phe Phe Gly Gly 130 - # 135 - # 140 - -
Asp Ser Val Gly Tyr Leu Asp Ile Ala Leu Gl - #y Ala Leu Val Ala Trp
145 1 - #50 1 - #55 1 - #60 - - Met Arg Ala Thr Glu Ala Arg His Gly
Leu Ar - #g Leu Phe Asp Ala Ser 165 - # 170 - # 175 - - Arg Val Arg
Cys Trp Arg Ser Gly Trp Ser Al - #a Ser Ala Ser Trp Thr 180 - # 185
- # 190 - - Arg Ser Trp Arg Ser Cys Arg Thr Ser Thr Gl - #y 195 - #
200 - - - - <210> SEQ ID NO 59 <211> LENGTH: 967
<212> TYPE: DNA <213> ORGANISM: maize - - <400>
SEQUENCE: 59 - - gggctagcta gtcttgcaga ctcggagata cgactagctt
gttataacaa gc - #caagcaga 60 - - gcggtgggga aaacaatggc gggcaatgag
ggtcttaagg tccttggcct gc - #aggtgagc 120 - - ccgttcgtgc tccgcgtgtg
catggcgttg aacacaaaag gagtgagcta cg - #agtacgtt 180 - - gaggaggacc
tatccaacaa gagtgagctc ctgcttaagt ccaacccggt gc - #acaagaag 240 - -
gtacccgtgc tcatccacaa cggtaagccc atctgtgagt cactcgtcat ca -
#tgcagtac 300 - - gtcgacgagc tgttcgccgg ccggtcgatc ctaccaaccg
acccctacga gc - #gcgccact 360 - - gctcgcttct gggctgccta cgccgacgac
aagttgttgc cagcgtggta cg - #gcatggtg 420 - - aaggcccagt cggcggagga
gagagcggag aaggtggagg agacgctttc cg - #cgatccag 480 - - cacatggaag
tggccttcgc caagtgctcc ggcggcaacg ccgccttctt cg - #gcggcgac 540 - -
tccattggct acgtcgacat cgtgctcggc tccttcttgt tctggttcga gg -
#cggtgcgc 600 - - agggtttacg acttggagat cattaacgct agcaatactc
cgctcttggc tg - #cgtgggcg 660 - - gagcggtttg tagggactgt agaagcaaag
gaggtggtgc cggtgcccga cg - #tggacatg 720 - - gccgtacagt gcatcaataa
gcttcatgcc cctgccgccg ccataagttc ac - #aatgagtc 780 - - gtgtaagtgt
aataaccagg aaaaggtaaa tggtgcggtg ctatggtcca aa - #ttccaacc 840 - -
gaataatgtt caaagcttac attgataggc ttgggttgtt gtcatcaaat aa -
#tgtggttc 900 - - agtcgtctcc tctgcaataa atataaatat gattgtttta
gtgtgtaaaa aa - #aaaaaaaa 960 - - aaaaaaa - # - # - # 967 - - - -
<210> SEQ ID NO 60 <211> LENGTH: 233 <212> TYPE:
PRT <213> ORGANISM: maize - - <400> SEQUENCE: 60 - -
Met Ala Gly Asn Glu Gly Leu Lys Val Leu Gl - #y Leu Gln Val Ser Pro
1 5 - # 10 - # 15 - - Phe Val Leu Arg Val Cys Met Ala Leu Asn Th -
#r Lys Gly Val Ser Tyr 20 - # 25 - # 30 - - Glu Tyr Val Glu Glu Asp
Leu Ser Asn Lys Se - #r Glu Leu Leu Leu Lys 35 - # 40 - # 45
- - Ser Asn Pro Val His Lys Lys Val Pro Val Le - #u Ile His Asn Gly
Lys 50 - # 55 - # 60 - - Pro Ile Cys Glu Ser Leu Val Ile Met Gln Ty
- #r Val Asp Glu Leu Phe 65 - # 70 - # 75 - # 80 - - Ala Gly Arg
Ser Ile Leu Pro Thr Asp Pro Ty - #r Glu Arg Ala Thr Ala 85 - # 90 -
# 95 - - Arg Phe Trp Ala Ala Tyr Ala Asp Asp Lys Le - #u Leu Pro
Ala Trp Tyr 100 - # 105 - # 110 - - Gly Met Val Lys Ala Gln Ser Ala
Glu Glu Ar - #g Ala Glu Lys Val Glu 115 - # 120 - # 125 - - Glu Thr
Leu Ser Ala Ile Gln His Met Glu Va - #l Ala Phe Ala Lys Cys 130 - #
135 - # 140 - - Ser Gly Gly Asn Ala Ala Phe Phe Gly Gly As - #p Ser
Ile Gly Tyr Val 145 1 - #50 1 - #55 1 - #60 - - Asp Ile Val Leu Gly
Ser Phe Leu Phe Trp Ph - #e Glu Ala Val Arg Arg 165 - # 170 - # 175
- - Val Tyr Asp Leu Glu Ile Ile Asn Ala Ser As - #n Thr Pro Leu Leu
Ala 180 - # 185 - # 190 - - Ala Trp Ala Glu Arg Phe Val Gly Thr Val
Gl - #u Ala Lys Glu Val Val 195 - # 200 - # 205 - - Pro Val Pro Asp
Val Asp Met Ala Val Gln Cy - #s Ile Asn Lys Leu His 210 - # 215 - #
220 - - Ala Pro Ala Ala Ala Ile Ser Ser Gln 225 2 - #30 - - - -
<210> SEQ ID NO 61 <211> LENGTH: 900 <212> TYPE:
DNA <213> ORGANISM: maize - - <400> SEQUENCE: 61 - -
ggccaagaac tcgatccgag caaaaaaatg tcggaggccg ccgtgcgagt ga -
#tcggccta 60 - - tggccgagcc cgttcgtgat ccgcgtcctg atcgcgctga
agctgaagca tg - #tggagtac 120 - - gagttcgtgg aggaggtggt gggcagcaag
agcgagctgc tgctcgcgtc ga - #acccggtg 180 - - cacaagaaga tccccgtcct
gctccaccac ggcaagcccc tctccgagtc cc - #taatcatc 240 - - gttcagtaca
tcgacgaggt ctggtcctcc ggcgcgccgg cggccatcct cc - #ccgccgac 300 - -
ccttacgcgc gcgctgtcca gcggttctgg gcgcagtacg tcgacgacaa ga -
#tgcacccg 360 - - gcgatccgcg tactgaaggg aacgtacgac ggggacaagg
agcaggcggc gg - #ggcagctg 420 - - tccgcggccc tgcagctcct ggaggaggct
ttcgcgcagc tcggccaggg ga - #agcgctac 480 - - ttcggcgggg acagcgtcgg
gtacctggac atcgccctgg tgtcgcacgt cg - #gctgggtg 540 - - aaggcggtgg
agaagatcgc gggggtcact ctgctggacg aggcgaaggt tc - #ccaacctg 600 - -
gtggcgtggg ctgaccggct gtgcgcccac ccggccgtgg tggacgcgat cc -
#ctgacgcc 660 - - gacaagttcg ttgagttcag cgtgacctat gggtcgttct
cttaatccta tc - #aatgctcc 720 - - caaagtgagc aaaatggctc cgcattgcgc
tttgtgattt tcactgcgca tg - #tgccggtg 780 - - catgtcaccg tcaatagcat
gtagtttgta tacatgtcct tctgtgaaaa ta - #aagctttg 840 - - ctgcccgtca
aagggaaatc gacataaaaa aaaaaaaaaa aaaaaaaaaa aa - #aaaaaaaa 900 - -
- - <210> SEQ ID NO 62 <211> LENGTH: 225 <212>
TYPE: PRT <213> ORGANISM: maize - - <400> SEQUENCE: 62
- - Met Ser Glu Ala Ala Val Arg Val Ile Gly Le - #u Trp Pro Ser Pro
Phe 1 5 - # 10 - # 15 - - Val Ile Arg Val Leu Ile Ala Leu Lys Leu
Ly - #s His Val Glu Tyr Glu 20 - # 25 - # 30 - - Phe Val Glu Glu
Val Val Gly Ser Lys Ser Gl - #u Leu Leu Leu Ala Ser 35 - # 40 - #
45 - - Asn Pro Val His Lys Lys Ile Pro Val Leu Le - #u His His Gly
Lys Pro 50 - # 55 - # 60 - - Leu Ser Glu Ser Leu Ile Ile Val Gln
Tyr Il - #e Asp Glu Val Trp Ser 65 - # 70 - # 75 - # 80 - - Ser Gly
Ala Pro Ala Ala Ile Leu Pro Ala As - #p Pro Tyr Ala Arg Ala 85 - #
90 - # 95 - - Val Gln Arg Phe Trp Ala Gln Tyr Val Asp As - #p Lys
Met His Pro Ala 100 - # 105 - # 110 - - Ile Arg Val Leu Lys Gly Thr
Tyr Asp Gly As - #p Lys Glu Gln Ala Ala 115 - # 120 - # 125 - - Gly
Gln Leu Ser Ala Ala Leu Gln Leu Leu Gl - #u Glu Ala Phe Ala Gln 130
- # 135 - # 140 - - Leu Gly Gln Gly Lys Arg Tyr Phe Gly Gly As - #p
Ser Val Gly Tyr Leu 145 1 - #50 1 - #55 1 - #60 - - Asp Ile Ala Leu
Val Ser His Val Gly Trp Va - #l Lys Ala Val Glu Lys 165 - # 170 - #
175 - - Ile Ala Gly Val Thr Leu Leu Asp Glu Ala Ly - #s Val Pro Asn
Leu Val 180 - # 185 - # 190 - - Ala Trp Ala Asp Arg Leu Cys Ala His
Pro Al - #a Val Val Asp Ala Ile 195 - # 200 - # 205 - - Pro Asp Ala
Asp Lys Phe Val Glu Phe Ser Va - #l Thr Tyr Gly Ser Phe 210 - # 215
- # 220 - - Ser 225 - - - - <210> SEQ ID NO 63 <211>
LENGTH: 872 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 63 - - ggaaacaaag aaataaagag agacgatggg
cggagaagaa ggcggcgacg gg - #ctgaagct 60 - - gatcgggcag tacgggagcg
cgttcgtgac gagggtgaag cttgctctca gc - #ctcaaggg 120 - - gctgagctac
gagtacgtcg aggaggatct cagaaacaag agcgcgctcc tc - #ctcagctc 180 - -
caacccggtg cacaaggcgg ttccagtgct gatccacaga ggcaagccta tc -
#tgcgagtc 240 - - gcaggtcatc gtgcagtaca tcgacgaggc ctttgccggc
atcggcccgc cc - #ctcctccc 300 - - ggccgacccc tacgaacgct cggtggcccg
tttctgggct gccttcattg aa - #gacaagct 360 - - tgtgtccccg tgggaccgag
tgttccgggc gaagacggag gacgagaggg aa - #gaggcgat 420 - - gaagcagatg
cttgcggcag tggacgctct ggagggagca ctgaaggagg gg - #agacccag 480 - -
acccttcttc ggcggcgaca gcgtcgggta cgtggacgtc gttctgggcg gt -
#gccgtctc 540 - - gtacgccaag gggcacgacg cgctcttcgg ttccgagctc
atcgacgccg cc - #aagacgcc 600 - - gctcctggcc gcgtggatgg agcgcttctg
cgagctcgac gcggccaagg cg - #gtcctgca 660 - - ggacgtcgat agagtggtcc
agtacggcaa gatgctgatc gccaagaatg ct - #gctgccac 720 - - tcgtcaggcg
tagtgttttt ctgatcgatc agcttgtatg tatatgaatt ga - #acttgtaa 780 - -
aaccaaatcg ttcaagtttg atggtaagtt ccatgttaga aaaaaaaaaa aa -
#aaaaaaaa 840 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa - # - # 872 -
- - - <210> SEQ ID NO 64 <211> LENGTH: 235 <212>
TYPE: PRT <213> ORGANISM: maize - - <400> SEQUENCE: 64
- - Met Gly Gly Glu Glu Gly Gly Asp Gly Leu Ly - #s Leu Ile Gly Gln
Tyr 1 5 - # 10 - # 15 - - Gly Ser Ala Phe Val Thr Arg Val Lys Leu
Al - #a Leu Ser Leu Lys Gly 20 - # 25 - # 30 - - Leu Ser Tyr Glu
Tyr Val Glu Glu Asp Leu Ar - #g Asn Lys Ser Ala Leu 35 - # 40 - #
45 - - Leu Leu Ser Ser Asn Pro Val His Lys Ala Va - #l Pro Val Leu
Ile His 50 - # 55 - # 60 - - Arg Gly Lys Pro Ile Cys Glu Ser Gln
Val Il - #e Val Gln Tyr Ile Asp 65 - # 70 - # 75 - # 80 - - Glu Ala
Phe Ala Gly Ile Gly Pro Pro Leu Le - #u Pro Ala Asp Pro Tyr 85 - #
90 - # 95 - - Glu Arg Ser Val Ala Arg Phe Trp Ala Ala Ph - #e Ile
Glu Asp Lys Leu 100 - # 105 - # 110 - - Val Ser Pro Trp Asp Arg Val
Phe Arg Ala Ly - #s Thr Glu Asp Glu Arg 115 - # 120 - # 125 - - Glu
Glu Ala Met Lys Gln Met Leu Ala Ala Va - #l Asp Ala Leu Glu Gly 130
- # 135 - # 140 - - Ala Leu Lys Glu Gly Arg Pro Arg Pro Phe Ph - #e
Gly Gly Asp Ser Val 145 1 - #50 1 - #55 1 - #60 - - Gly Tyr Val Asp
Val Val Leu Gly Gly Ala Va - #l Ser Tyr Ala Lys Gly 165 - # 170 - #
175 - - His Asp Ala Leu Phe Gly Ser Glu Leu Ile As - #p Ala Ala Lys
Thr Pro 180 - # 185 - # 190 - - Leu Leu Ala Ala Trp Met Glu Arg Phe
Cys Gl - #u Leu Asp Ala Ala Lys 195 - # 200 - # 205 - - Ala Val Leu
Gln Asp Val Asp Arg Val Val Gl - #n Tyr Gly Lys Met Leu 210 - # 215
- # 220 - - Ile Ala Lys Asn Ala Ala Ala Thr Arg Gln Al - #a 225 2 -
#30 2 - #35 - - - - <210> SEQ ID NO 65 <211> LENGTH:
971 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 65 - - gtgactgtga tctatactat aaggtgaaca
agatctcttt gtctactgta gt - #tgcagcac 60 - - cagcagcagc agcagaagag
cagcgcctga gctccagcaa taatggccga ga - #agggcgtg 120 - - aaggtgttgg
ggatgtgggc gagccccatg gtgatcaggg tggagtgggc gc - #tgcggctg 180 - -
aagggcgtcg agtacgagta cgtcgacgag gacctcgcca acaagagcgc cg -
#acctgctc 240 - - cgccacaacc cggtgaccaa gaaggtgccc gtgctcgtcc
acgacggcaa gc - #cggtcgcg 300 - - gagtccacca tcatcgtgga gtacatcgac
gaggtctgga agggcggcta cc - #ccatcatg 360 - - ccgggcgacc cctacgagcg
cgcccaggcg aggttctggg ccaggttcgc gg - #aagacaag 420 - - tgcaacgctg
ctctgtaccc gatcttcacc gcgaccggcg aggcgcagcg ca - #aggcggtg 480 - -
cacgaggccc agcagtgcct caagaccctg gagacggcct tggaggggaa ga -
#agttcttc 540 - - ggcggcgacg ccgtgggcta cctcgacatc gtcgtcgggt
ggttcgcgca ct - #ggctcccc 600 - - gtcatcgagg aggtgaccgg cgccagcgtc
gtcacccacg aggagctgcc gc - #tgatgaag 660 - - gcctggttcg gtcggttcct
cgcccttgac gtggtgaagg cggccctgcc cg - #acagggac 720 - - aggctcctcg
ccgccaacaa ggcccgccgt gagcagctcc tctccgcgta ga - #tatggcta 780 - -
gtaattctgg agcagctagt ttcaccgccg acgctcatat attgctgaat aa -
#ggactggt 840 - - tgcacttttg cacgttgtgc agtgcagccc gaggtttgga
tgacctctgc cc - #ctctgttc 900 - - catttcagaa tggtagtccc ataataatgc
atatacatca tgcaaaaaaa aa - #aaaaaaaa 960 - - aaaaaaaaaa a - # - # -
# 971 - - - - <210> SEQ ID NO 66 <211> LENGTH: 222
<212> TYPE: PRT <213> ORGANISM: maize - - <400>
SEQUENCE: 66 - - Met Ala Glu Lys Gly Val Lys Val Leu Gly Me - #t
Trp Ala Ser Pro Met 1 5 - # 10 - # 15 - - Val Ile Arg Val Glu Trp
Ala Leu Arg Leu Ly - #s Gly Val Glu Tyr Glu 20 - # 25 - # 30 - -
Tyr Val Asp Glu Asp Leu Ala Asn Lys Ser Al - #a Asp Leu Leu Arg His
35 - # 40 - # 45 - - Asn Pro Val Thr Lys Lys Val Pro Val Leu Va -
#l His Asp Gly Lys Pro 50 - # 55 - # 60 - - Val Ala Glu Ser Thr Ile
Ile Val Glu Tyr Il - #e Asp Glu Val Trp Lys 65 - # 70 - # 75 - # 80
- - Gly Gly Tyr Pro Ile Met Pro Gly Asp Pro Ty - #r Glu Arg Ala Gln
Ala 85 - # 90 - # 95 - - Arg Phe Trp Ala Arg Phe Ala Glu Asp Lys Cy
- #s Asn Ala Ala Leu Tyr 100 - # 105 - # 110 - - Pro Ile Phe Thr
Ala Thr Gly Glu Ala Gln Ar - #g Lys Ala Val His Glu 115 - # 120 - #
125 - - Ala Gln Gln Cys Leu Lys Thr Leu Glu Thr Al - #a Leu Glu Gly
Lys Lys 130 - # 135 - # 140 - - Phe Phe Gly Gly Asp Ala Val Gly Tyr
Leu As - #p Ile Val Val Gly Trp 145 1 - #50 1 - #55 1 - #60 - - Phe
Ala His Trp Leu Pro Val Ile Glu Glu Va - #l Thr Gly Ala Ser Val 165
- # 170 - # 175 - - Val Thr His Glu Glu Leu Pro Leu Met Lys Al - #a
Trp Phe Gly Arg Phe 180 - # 185 - # 190 - - Leu Ala Leu Asp Val Val
Lys Ala Ala Leu Pr - #o Asp Arg Asp Arg Leu 195 - # 200 - # 205 - -
Leu Ala Ala Asn Lys Ala Arg Arg Glu Gln Le - #u Leu Ser Ala 210 - #
215 - # 220 - - - - <210> SEQ ID NO 67 <211> LENGTH:
1074 <212> TYPE: DNA <213> ORGANISM: maize - -
<400> SEQUENCE: 67 - - gctctaacac agcgcaagcc atggcaggac
gagtagcgga caaagaccca ga -
#gctgaagg 60 - - tgctcggagt gtggtcgagc ccgttcgtta tcagggcccg
cgtcgcgcta aa - #cctcaagg 120 - - gcctggccta ccgatacgtg gaggacaacc
tggacagcaa gagcgagctc ct - #cctcgcct 180 - - ccaaccccgt gcacgggaag
gtgccggtgc tcctccacga cggcaggccc gt - #ctgcgagt 240 - - cccgggtcat
cgtggagtat atcgacgagg ccttcccggc cagcggcccc tg - #cctcctcc 300 - -
ccgccgaccc gtaccgccgc gccgtcgacc gcttctgggc ctcctacgcc ga -
#cgacaagc 360 - - tctttcccac ctggataccc gtctacaacg gcaggacgag
cgaggacagg gt - #cgcggcgg 420 - - cgaggcaggt cgtggccgtg ctggagaagt
ttgagcaggc gttcgatgag tg - #ctccgggt 480 - - ccgggggcaa ggcgttcttc
ggcggggacg ctgctggcct cgtggacgtc gt - #gctaggcg 540 - - gcttcctcgg
gtggctgcgc gcgtctgagg cgatgtgtgg cgtgagggtc at - #cgaccccg 600 - -
ccaagacgcc gctgctggcg gcgtgggcgg accggttcgc cgcgctcgac gg -
#cgtcaggg 660 - - aggtgatacc tgacgtgcag aggctgctgg agtataacaa
gattaggcga gc - #tcgtcgtg 720 - - ggctgccgta ggtgctgggc cttgggccat
ctatctgtca ccatgtggtc ag - #tcaactct 780 - - aagcaggaga ctttgacaag
gttgaaagtt agttcatgaa ggtgggcaat ct - #aattagga 840 - - tgctcatgct
ttagctagga gtgccattag ttttcctgtt gaaaggcatg tt - #tggttcct 900 - -
ctttcctact gaaagagttt gtaatataat ctatgatgct ttgagtttga ga -
#aaagaact 960 - - atgaataaaa catggatatc ttccgatatt tcagttcaaa
ttaaaaaaaa aa - #aaaaaaaa 1020 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa - #aa 1074 - - - - <210> SEQ ID NO
68 <211> LENGTH: 236 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 68 - - Met Ala Gly Arg
Val Ala Asp Lys Asp Pro Gl - #u Leu Lys Val Leu Gly 1 5 - # 10 - #
15 - - Val Trp Ser Ser Pro Phe Val Ile Arg Ala Ar - #g Val Ala Leu
Asn Leu 20 - # 25 - # 30 - - Lys Gly Leu Ala Tyr Arg Tyr Val Glu
Asp As - #n Leu Asp Ser Lys Ser 35 - # 40 - # 45 - - Glu Leu Leu
Leu Ala Ser Asn Pro Val His Gl - #y Lys Val Pro Val Leu 50 - # 55 -
# 60 - - Leu His Asp Gly Arg Pro Val Cys Glu Ser Ar - #g Val Ile
Val Glu Tyr 65 - # 70 - # 75 - # 80 - - Ile Asp Glu Ala Phe Pro Ala
Ser Gly Pro Cy - #s Leu Leu Pro Ala Asp 85 - # 90 - # 95 - - Pro
Tyr Arg Arg Ala Val Asp Arg Phe Trp Al - #a Ser Tyr Ala Asp Asp 100
- # 105 - # 110 - - Lys Leu Phe Pro Thr Trp Ile Pro Val Tyr As - #n
Gly Arg Thr Ser Glu 115 - # 120 - # 125 - - Asp Arg Val Ala Ala Ala
Arg Gln Val Val Al - #a Val Leu Glu Lys Phe 130 - # 135 - # 140 - -
Glu Gln Ala Phe Asp Glu Cys Ser Gly Ser Gl - #y Gly Lys Ala Phe Phe
145 1 - #50 1 - #55 1 - #60 - - Gly Gly Asp Ala Ala Gly Leu Val Asp
Val Va - #l Leu Gly Gly Phe Leu 165 - # 170 - # 175 - - Gly Trp Leu
Arg Ala Ser Glu Ala Met Cys Gl - #y Val Arg Val Ile Asp 180 - # 185
- # 190 - - Pro Ala Lys Thr Pro Leu Leu Ala Ala Trp Al - #a Asp Arg
Phe Ala Ala 195 - # 200 - # 205 - - Leu Asp Gly Val Arg Glu Val Ile
Pro Asp Va - #l Gln Arg Leu Leu Glu 210 - # 215 - # 220 - - Tyr Asn
Lys Ile Arg Arg Ala Arg Arg Gly Le - #u Pro 225 2 - #30 2 - #35 - -
- - <210> SEQ ID NO 69 <211> LENGTH: 904 <212>
TYPE: DNA <213> ORGANISM: maize - - <400> SEQUENCE: 69
- - gacaccacag gcataacaga acagaaccat cgatcgagct tggttgctcg gc -
#agcagcta 60 - - gcaatggccg ccaatggagg tgatgagctg aagctgctgg
gcgtgtggga ca - #gcccgtac 120 - - gtcaacaggg tccagatcgt gctcaacctc
aagggcctca gctacgagta cg - #tggaggag 180 - - gacctcctca gcaagagcga
gctcctcctc aattccaacc cggtgcacaa ga - #aagtgccc 240 - - gtgctcatcc
acgccggcaa gccggtcgcc gagtcgcagg ccatcgttca gt - #acctcgac 300 - -
gaggctttcc ccagcggcac gttcccgtcg gtcctcccag ccgaacccta cg -
#cacgcgcc 360 - - accgcccgct tctgggccgc cttcgtcgac gacaaggtcg
ggtctccatg gc - #acacggtc 420 - - ctgttcgcgc gggagcacgg gaagaaggcg
gacgcggcgt cgcggatcgt cg - #cggcgctg 480 - - gagacgctgg agggtgcgtt
cgaggactgc tccggcggga gggactactt cg - #gcggcgac 540 - - gccatcggct
tcgtggacgt ggtcctcggc agctacctgg gctggttcaa gg - #tgttcgag 600 - -
aagatggtcg gcgtcagggt cctggacgtg gcgaggacgc cgctcctcgc cg -
#cgtggggg 660 - - gagcgtttcg cggcggcgga agcggccaag gacgtcctgc
cggatgacgt tg - #acaaggtg 720 - - ctcgagttcc ttcagaagtt cctggattag
atgcgcgcca ccatgtgctc cg - #gtgtccaa 780 - - ctcccaatgt ttgtttgctt
tggtcatttt cggtgcgctg ttaatgggcc tc - #ggatgttt 840 - - gccagttgat
taacttgatt ttatagaatc ttaataatat tctaaaacaa aa - #aaaaaaaa 900 - -
aaaa - # - # - # 904 - - - - <210> SEQ ID NO 70 <211>
LENGTH: 228 <212> TYPE: PRT <213> ORGANISM: maize - -
<400> SEQUENCE: 70 - - Met Ala Ala Asn Gly Gly Asp Glu Leu
Lys Le - #u Leu Gly Val Trp Asp 1 5 - # 10 - # 15 - - Ser Pro Tyr
Val Asn Arg Val Gln Ile Val Le - #u Asn Leu Lys Gly Leu 20 - # 25 -
# 30 - - Ser Tyr Glu Tyr Val Glu Glu Asp Leu Leu Se - #r Lys Ser
Glu Leu Leu 35 - # 40 - # 45 - - Leu Asn Ser Asn Pro Val His Lys
Lys Val Pr - #o Val Leu Ile His Ala 50 - # 55 - # 60 - - Gly Lys
Pro Val Ala Glu Ser Gln Ala Ile Va - #l Gln Tyr Leu Asp Glu 65 - #
70 - # 75 - # 80 - - Ala Phe Pro Ser Gly Thr Phe Pro Ser Val Le -
#u Pro Ala Glu Pro Tyr 85 - # 90 - # 95 - - Ala Arg Ala Thr Ala Arg
Phe Trp Ala Ala Ph - #e Val Asp Asp Lys Val 100 - # 105 - # 110 - -
Gly Ser Pro Trp His Thr Val Leu Phe Ala Ar - #g Glu His Gly Lys Lys
115 - # 120 - # 125 - - Ala Asp Ala Ala Ser Arg Ile Val Ala Ala Le
- #u Glu Thr Leu Glu Gly 130 - # 135 - # 140 - - Ala Phe Glu Asp
Cys Ser Gly Gly Arg Asp Ty - #r Phe Gly Gly Asp Ala 145 1 - #50 1 -
#55 1 - #60 - - Ile Gly Phe Val Asp Val Val Leu Gly Ser Ty - #r Leu
Gly Trp Phe Lys 165 - # 170 - # 175 - - Val Phe Glu Lys Met Val Gly
Val Arg Val Le - #u Asp Val Ala Arg Thr 180 - # 185 - # 190 - - Pro
Leu Leu Ala Ala Trp Gly Glu Arg Phe Al - #a Ala Ala Glu Ala Ala 195
- # 200 - # 205 - - Lys Asp Val Leu Pro Asp Asp Val Asp Lys Va - #l
Leu Glu Phe Leu Gln 210 - # 215 - # 220 - - Lys Phe Leu Asp 225 - -
- - <210> SEQ ID NO 71 <211> LENGTH: 1013 <212>
TYPE: DNA <213> ORGANISM: maize - - <400> SEQUENCE: 71
- - ctggtctctt gcacagactc ggagcaagat aggcctagct ggttacaagc ca -
#agaacaag 60 - - tagagcggta gagggaaaat atatctggag agggaaacaa
tggcgggcga gg - #agggtctt 120 - - aaggtcctcg gcctgcaggt gagcccgttc
gtgctccgcg tgtgcttggc gc - #tgaacatg 180 - - aaaggagtga gttacgagta
cgtcgaggag gacatatcca acaagagtga gc - #tcctgctc 240 - - aagtccaacc
cggtgcacaa gaaggtgccc gtgctcatcc acaacggtaa gc - #ccatctgc 300 - -
gagtcactcg tcatcatgca gtacgtcgac gagctgttcg ccggccggcc ga -
#tcctccca 360 - - accgacccct acgagcgcgc cactgctcgc ttctgggctg
cctacgccga cg - #acaagttg 420 - - tttccagcgt ggtacggcat ggtgaaggcc
cagccggagg aggagagggc gg - #agaaggcg 480 - - aaggagacgc tcgccgccat
cgagcacatg gaagtgacct tcgccaagtg ct - #ccggcggc 540 - - aacgccttct
tcggtggcga ctccatcggc tacgtcgaca tcgtgctgac gt - #gctcggct 600 - -
ccttcttgtt ctggttcgag gcggtgcgca gggttttcga cctggagatc at -
#taacgcta 660 - - gcaagactcc gctgttggct gcgtgggcgg agcggtttgt
agggactgta ga - #agcgaagg 720 - - aggtggtgcc gttgcccacg gcggacatgg
cggtacagta catcaataag ct - #tcatgccc 780 - - ccctgccgcc gccatgagtt
cacaatgagt cgtgtaagtg taaccaagca gg - #gaaaaagg 840 - - taaatggtgc
ggtgctttgg tccaaattcc aaccgaataa tgttcaaagc tt - #atattgat 900 - -
aggcttgggt tgttgtcatc aaatattgtg gttcagtcgt ctcctctgca at -
#aaatataa 960 - - atatgattat tactttcttt gccgtaaaaa aaaaaaaaaa
aaaaaaaaaa aa - #a 1013 - - - - <210> SEQ ID NO 72
<211> LENGTH: 200 <212> TYPE: PRT <213> ORGANISM:
maize - - <400> SEQUENCE: 72 - - Met Ala Gly Glu Glu Gly Leu
Lys Val Leu Gl - #y Leu Gln Val Ser Pro 1 5 - # 10 - # 15 - - Phe
Val Leu Arg Val Cys Leu Ala Leu Asn Me - #t Lys Gly Val Ser Tyr 20
- # 25 - # 30 - - Glu Tyr Val Glu Glu Asp Ile Ser Asn Lys Se - #r
Glu Leu Leu Leu Lys 35 - # 40 - # 45 - - Ser Asn Pro Val His Lys
Lys Val Pro Val Le - #u Ile His Asn Gly Lys 50 - # 55 - # 60 - -
Pro Ile Cys Glu Ser Leu Val Ile Met Gln Ty - #r Val Asp Glu Leu Phe
65 - # 70 - # 75 - # 80 - - Ala Gly Arg Pro Ile Leu Pro Thr Asp Pro
Ty - #r Glu Arg Ala Thr Ala 85 - # 90 - # 95 - - Arg Xaa Trp Ala
Ala Tyr Ala Asp Asp Lys Le - #u Phe Pro Ala Trp Tyr 100 - # 105 - #
110 - - Gly Met Val Lys Ala Gln Pro Glu Glu Glu Ar - #g Ala Glu Lys
Ala Lys 115 - # 120 - # 125 - - Glu Thr Leu Ala Ala Ile Glu His Met
Glu Va - #l Thr Phe Ala Lys Cys 130 - # 135 - # 140 - - Ser Gly Gly
Asn Ala Phe Phe Gly Gly Asp Se - #r Ile Gly Tyr Val Asp 145 1 - #50
1 - #55 1 - #60 - - Ile Val Leu Thr Cys Ser Ala Pro Ser Cys Se - #r
Gly Ser Arg Arg Cys 165 - # 170 - # 175 - - Ala Gly Phe Ser Thr Trp
Arg Ser Leu Thr Le - #u Ala Arg Leu Arg Cys 180 - # 185 - # 190 - -
Trp Leu Arg Gly Arg Ser Gly Leu 195 - # 200 - - - - <210> SEQ
ID NO 73 <211> LENGTH: 1068 <212> TYPE: DNA <213>
ORGANISM: maize - - <400> SEQUENCE: 73 - - ggcagcacaa
acgagcacaa taatggccgg aggaggtgac gatgaactca ag - #ctgctggg 60 - -
ggcgtgggcg agcccattcg tcctgcgggt gaagctcgcg ctcagcttca ag -
#ggcctgag 120 - - ctacgaggac gtggaggagg acctctccgg cggcaagagc
gagctgctcc tc - #gagtccaa 180 - - cccggtgcac aagaaggtgc ccgtgctcct
ccacaacggc aagcctgtgt gc - #gagtcgca 240 - - gatcatcgtg cagtacatcg
atgaggcctt cgccggcact ggcccgtccc tt - #ctccctgc 300 - - cgacccgcac
cagcgcgccg tggctcgctt ctggggtgcc tacattgacg ac - #aagctcct 360 - -
agccttctgg ctgcaatcag caagggccaa gacgcaggag gaaaaggccg ag -
#gcgctgaa 420 - - gcaggcgctc gccgcggccg agaacctgga ggccgccttc
acggagatct cc - #gagggcaa 480 - - gcccttcttc ggcggcgaca gcgtcgggta
cctggacgtg acgctgggag cg - #ctggtcgc 540 - - gtgggtgcac gccgccgaga
agctgtacgg gatgaggctc ttcgacgcca cg - #aggacccc 600 - - gcggctgagc
gcgttcgtgg agaggttcgg cgcgctcgga gcggccaagg cg - #gtgctgcc 660 - -
cgacgtcgat ggcctcgtcg aatacgccaa acagaggcag gccgacgcgg ca -
#gctgcagc 720 - - ctcggacagc taaaaaaatg gcaccgcgag tttaccgacg
tacggcagtc ag - #tgctggac 780 - - gaagcaagat tatgggtatt ctgcatatac
tattcagctg ctgtcgtgtg ta - #ttagctgg 840
- - ttgttactag attgttggcg tgtgacaaag aaaataaaaa tggatgggcc gg -
#ctttcgtt 900 - - tgtgtttgta ttgtacgttt gccgtttggt gtgtaccgtg
tgtcgtaggt cg - #gaaattgc 960 - - cgcatatcgg catgcctagt gtaaccctgt
cgattatgca gtctggtttg ct - #ttatattc 1020 - - accaaagtaa gtaatctgaa
taattttctt gaaaaaaaaa aaaaaaaa - # 1068 - - - - <210> SEQ ID
NO 74 <211> LENGTH: 236 <212> TYPE: PRT <213>
ORGANISM: maize - - <400> SEQUENCE: 74 - - Met Ala Gly Gly
Gly Asp Asp Glu Leu Lys Le - #u Leu Gly Ala Trp Ala 1 5 - # 10 - #
15 - - Ser Pro Phe Val Leu Arg Val Lys Leu Ala Le - #u Ser Phe Lys
Gly Leu 20 - # 25 - # 30 - - Ser Tyr Glu Asp Val Glu Glu Asp Leu
Ser Gl - #y Gly Lys Ser Glu Leu 35 - # 40 - # 45 - - Leu Leu Glu
Ser Asn Pro Val His Lys Lys Va - #l Pro Val Leu Leu His 50 - # 55 -
# 60 - - Asn Gly Lys Pro Val Cys Glu Ser Gln Ile Il - #e Val Gln
Tyr Ile Asp 65 - # 70 - # 75 - # 80 - - Glu Ala Phe Ala Gly Thr Gly
Pro Ser Leu Le - #u Pro Ala Asp Pro His 85 - # 90 - # 95 - - Gln
Arg Ala Val Ala Arg Phe Trp Gly Ala Ty - #r Ile Asp Asp Lys Leu 100
- # 105 - # 110 - - Leu Ala Phe Trp Leu Gln Ser Ala Arg Ala Ly - #s
Thr Gln Glu Glu Lys 115 - # 120 - # 125 - - Ala Glu Ala Leu Lys Gln
Ala Leu Ala Ala Al - #a Glu Asn Leu Glu Ala 130 - # 135 - # 140 - -
Ala Phe Thr Glu Ile Ser Glu Gly Lys Pro Ph - #e Phe Gly Gly Asp Ser
145 1 - #50 1 - #55 1 - #60 - - Val Gly Tyr Leu Asp Val Thr Leu Gly
Ala Le - #u Val Ala Trp Val His 165 - # 170 - # 175 - - Ala Ala Glu
Lys Leu Tyr Gly Met Arg Leu Ph - #e Asp Ala Thr Arg Thr 180 - # 185
- # 190 - - Pro Arg Leu Ser Ala Phe Val Glu Arg Phe Gl - #y Ala Leu
Gly Ala Ala 195 - # 200 - # 205 - - Lys Ala Val Leu Pro Asp Val Asp
Gly Leu Va - #l Glu Tyr Ala Lys Gln 210 - # 215 - # 220 - - Arg Gln
Ala Asp Ala Ala Ala Ala Ala Ser As - #p Ser 225 2 - #30 2 - #35
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References